Methods, compositions, and kits for the selective activation of protoxins through combinatorial targeting

ABSTRACT

The present invention provides methods and compositions for treating various diseases through selective killing of targeted cells using a combinatorial targeting approach. The invention features protoxin fusion proteins containing a cell targeting moiety and, a modifiable activation moiety which is activated by an activation moiety not naturally operably found in, on, or in the vicinity of a target cell. These methods also include the combinatorial use of two or more therapeutic agents, at minimum comprising a protoxin and a protoxin activator, to target and destroy a specific cell population.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the U.S. National Stage of International Application No. PCT/US2007/16475, filed Jul. 20, 2007, which in turn, claims the benefit of U.S. Provisional Application No. 60/832,022, filed Jul. 20, 2006, each of which is incorporated by reference.

FIELD OF THE INVENTION

In general, the present invention relates to a therapeutic strategy for targeting cyotoxic or cytostatic agents to particular cell types while reducing systemic adverse effects. More specifically, the present invention involves the use of a therapeutic modality comprising two or more individually inactive components with independent targeting principles, which are activated through their specific interaction at the targeted cells. The invention also provides related methods and compositions.

BACKGROUND OF THE INVENTION

Selective killing of particular types of cells is desirable in a variety of clinical settings, including the treatment of cancer, which is usually manifested through growth and accumulation of malignant cells. An established treatment for cancer is chemotherapy, which kills tumor cells by inhibiting DNA synthesis or damaging DNA (Chabner and Roberts, Nat. Rev. Cancer 5:65 (2005)). However, such treatments often cause severe systemic toxicity due to nondiscriminatory killing of normal cells. Because many cancer chemotherapeutics exert their efficacy through selective destruction of proliferating cells, increased toxicities to normal tissues with high proliferation rates, such as bone marrow, gastrointestinal tract, and hair follicles have usually prevented their use in optimal doses. Such treatments often fail, resulting in drug resistance, disease relapse, and/or metastasis. To reduce systemic toxicity, different strategies have been explored to selectively target a particular cell population. Antibodies and other ligands that recognize tumor-associated antigens have been coupled with small molecule drugs or protein toxins, generating conjugates and fusion proteins that are often referred to as immunoconjugates and immunotoxins, respectively (Allen, Nat. Rev. Cancer 2:750 (2002)).

In addition to dose-limiting toxicities, another limitation for chemotherapy is its ineffectiveness for treatment of cancers that do not involve accelerated proliferation, but rather prolonged survival of malignant cells due to defective apoptosis (Kitada et al., Oncogene 21:3459 (2002)). For example, B cell chronic lymphocytic leukemia (B-CLL) is a disease characterized by slowly accumulating apoptosis-resistant neoplastic B cells, for which currently there is no cure (Munk and Reed, Leuk. Lymphoma 45:2365 (2004)).

Cancer stem cells (CSCs) are a small fraction of tumor cells that have a capacity for self-renewal and unlimited growth, and therefore are distinct from their progeny in their capacity to initiate cancers (Schulenburg et al., Cancer 107:2512 (2006)). Current cancer therapies do not target these cancer stem cells specifically, and it is hypothesized that the persistence of CSCs results in an ineradicable subset of cells that can give rise to progeny cells exhibiting drug resistance and/or contributing to the formation of metastases. In those tumors which harbor CSCs it is highly attractive to be able to eliminate these cells. CSCs have been thought to possess many properties similar to that of normal stems cells, e.g., long life span, relative mitotic quiescence, and active DNA repair capacity, as well as resistance to apoptosis and to drug/toxins through high level expression of ATP-binding cassette drug transporters such as P-glycoprotein. Consequently, CSCs are thought to be difficult to target and destroy by conventional cancer therapies (Dean et al., Nat. Rev. Cancer 5:275 (2005)). Conversely, it is critically important to distinguish CSCs from normal stem cells because of the essential roles that normal stem cells play in the renewal of normal tissues.

To increase the selectivity of highly toxic anti-tumor agents, various attempts have been made to take advantage of specific features of the tumor microenvironment, such as the low pH, low oxygen tension, or increased density of tumor specific enzymes, that are not found in the vicinity of normal cells in well-perfused tissues. Environmentally sensitive anti-tumor agents have been developed that are hypothesized to exhibit increased toxicity in the solid tumor. For example “bioreductive prodrugs” are agents that can be activated to cytotoxic agents in the hypoxic environment of a solid tumor (Ahn and Brown, Front Biosci. May 1, 2007; 12:3483-501.) Similarly Kohchi et al. describe the synthesis of chemotherapeutic prodrugs that can be activated by membrane dipeptidases found in tumors (Bioorg Med Chem Lett. Apr. 15, 2007; 17(8):2241-5.) The use of selective antibody conjugated enzymes to alter the tumor microenvironment has also been explored by many groups. In the strategy known as antibody-directed enzyme prodrug therapy (ADEPT), enzymes conjugated to tumor-specific antibodies are intended to be delivered to the patient, followed by a chemotherapeutic agent that is inactive until subject to the action of the conjugated enzyme (see for example Bagshawe, Expert Rev Anticancer Ther. October 2006; 6(10):1421-31 or Rooseboome et al. Pharmacol Rev. March 2004; 56(1):53-102) To date the clinical advantages of these strategies remain undocumented and there remains a high interest in developing more selective and more potent agents that can show therapeutic utility.

SUMMARY OF THE INVENTION

In one aspect, the invention features a protoxin activator fusion protein including one or more cell-targeting moieties and a modification domain. In one embodiment of this aspect, the protoxin activator fusion protein can also include a natively activatable domain. In this embodiment, the modification domain is inactive prior to activation of the natively activatable domain. Desirably, the protoxin activator fusion protein is non-toxic to a target cell (e.g., the protoxin activator fusion protein has less than 10% of the cytotoxic or cytostatic activity of the combination of the protoxin activator fusion protein and the protoxin upon which the protoxin activator fusion protein acts).

In the above aspects, the modification domain can be a protease containing the catalytic domain of a human protease (desirably an exogenous human protease), or a non-human protease, including a viral protease (e.g., retroviral protease, a potyviral protease, a picornaviral protease, or a coronaviral protease). In a related aspect, the modification domain can be a phosphatase.

In another aspect, the invention features a protoxin fusion protein including one or more non-native cell-targeting moieties, a selectively modifiable activation domain, and a toxin domain (e.g., an activatable toxin domain). In this aspect, the modifiable activation domain may include a substrate for an exogenous enzyme.

In this aspect, the exogenous enzyme can be, for example, a protease or phosphatase. Examples of proteases include an exogenous human protease or a non-human (or non-mammalian) protease, including a viral protease (e.g., a retroviral protease, a potyviral protease, a picornaviral protease, or a coronaviral protease).

Also in this aspect, the activatable toxin domain can include an activatable pore forming toxin or an activatable enzymatic toxin. Examples of such domains include an AB toxin, a cyotoxic necrotizing factor toxin, a dermonecrotic toxin, and an activatable ADP-ribosylating toxin. Further examples include aerolysin, Vibrio cholerae exotoxin, Pseudomonas exotoxin, and diphtheria toxin.

In the above protoxin fusion proteins, the modifiable activation domain may further include a post-translational modification of a protease cleavage site. In this aspect, the modifiable activation domain can include a substrate for an enzyme (e.g., an exogenous enzyme).

In another aspect, the invention features a proactivator fusion protein including one or more non-native cell-targeting moieties, a selectively modifiable activation domain, and an activator domain. In this aspect, the modifiable activation domain may include a substrate for an enzyme (e.g., a protease or phosphatase). The modifiable activation domain may include a post-translational modification of a protease cleavage site or a substrate for an enzyme capable of removing a post-translational modification.

In this aspect, the protease may be an exogenous human protease, a non-human protease (e.g., a non-mammalian protease), or a viral protease.

Any of the above compositions can be formulated for administration to a subject (e.g., a human, dog, cat, monkey, horse, or rat) in order to kill a desired population of target cells.

In yet another aspect, the invention features a method of destroying or inhibiting a target cell (e.g., a human cell or a human cancer cell), by contacting the target cell with (i) a protoxin fusion protein including a first cell-targeting moiety, a selectively modifiable activation domain (e.g. a protease domain heterologous to the target cell), and a toxin domain; and (ii) a protoxin activator fusion protein including a second cell-targeting moiety and a modification domain. In this aspect, the first cell-targeting moiety of the protoxin fusion protein and the second cell-targeting moiety of the protoxin activator fusion protein each recognize and bind the target cell. Upon binding of both fusion proteins to the target cell, the modifiable activation moiety is selectively activated by the modification domain resulting in toxin activity; and thereby destroying or inhibiting the target cell. In a separate embodiment, absent the selective activation of the modifiable activation domain, the protoxin fusion protein is not natively activatable by the target cell or the environment surrounding the target cell, and wherein the selective activation of the modifiable activation domains renders the protoxin fusion protein natively activatable.

In a related aspect, the invention features a method of destroying or inhibiting a target cell in a subject, by administering to the subject (e.g., a human) (i) a protoxin fusion protein including a first cell-targeting moiety, a selectively modifiable activation domain, and a toxin domain; and (ii) a protoxin activator fusion protein including a second cell-targeting moiety, a natively activatable domain, and a modification domain. In this aspect the natively activatable domain becoming active upon administration of the protoxin activator fusion protein to the subject, whereby the activity of the natively activatable domain results in activation of the modification domain. In this aspect, the first cell-targeting domain of the protoxin fusion protein and the second cell-targeting domain of the protoxin activator fusion protein each recognize and bind the target cell and, upon binding of both fusion proteins to the target cell, the modifiable activation moiety is selectively activated by the modification domain resulting in toxin activity; and thereby destroying or inhibiting the target cell.

In the above-related aspects, the toxin domain can include an AB toxin, a cyotoxic necrotizing factor toxin, a dermonecrotic toxin, activatable pore forming toxin, activatable enzymatic toxin, and an activatable ADP-ribosylating toxin. Additional examples of toxin domains include Vibrio Cholerae exotoxin, aerolysin, a caspase, Ricin, Abrin, and Modeccin.

Also in the above-related aspects, the heterologous proteases can include an exogenous human protease (e.g., human granzyme B, including amino acids 21-247 of human granzyme B), a non-human protease, a non-mammalian protease, or a viral protease. In this aspect the selectively modifiable activation domain can be IEPD.

Also in the above-related aspects, the toxin domain can include Diphtheria toxin (e.g., amino-acids 1-389 of Diphtheria toxin), where the Diphtheria toxin furin cleavage site is replaced by a cleavage site of a protease heterologous to the target cell.

Also in the above-related aspects, the protoxin fusion protein can be contacted with the target cell prior to, simultaneous with, or after the protoxin activator fusion protein is contacted with the cell.

In yet another aspect, the invention features a kit having a (i) protoxin fusion protein and (ii) a protoxin activator fusion protein, and (iii) instructions for administering the two fusion proteins to a patient diagnosed with cancer.

In another related aspect, the invention features a kit having a (i) protoxin fusion protein and (ii) instructions for administering (i) with a protoxin activator fusion protein to a patient diagnosed with cancer.

In yet another related aspect, the invention features a kit having a (i) protoxin activator fusion protein and (ii) instructions for administering (i) with a protoxin fusion protein to a patient diagnosed with cancer.

In any of the forgoing aspects, the one or more of the fusion proteins can be modified by PEGylation, glycosylation, or both.

Also in any of the forgoing aspects, the one ore more cell-targeting domains or non-native cell-targeting domains can be a polypeptide, an antibody (e.g., an antibody, an antibody-like molecule, an antibody fragment, and a single antibody domain, including an anti-CD5 ScFv, anti-CD19 ScFv, and an anti-CD22 ScFv), a ligand for a receptor, a matrix fragment, a soluble receptor fragment, a cytokine, a soluable mediator, or an artificially diversified binding protein. The cell-targeting moiety may derived from a bacterial source (e.g., derived from a bacterial toxin). Alternatively, the cell targeting moiety can be a carbohydrate, a lipid, or a synthetic ligand.

Further, the cell-targeting domains or non-native cell targeting domains of the invention can recognize a cancer cell, a hematopoietic cell (e.g., a lymphocyte), or a nociceptive neuron.

As used herein in the specification, “a” or “an” may mean one or more; “another” may mean at least a second or more.

The term “polypeptide” or “peptide” as used herein refers to two or more amino acids linked by an amide bond between the carboxyl terminus of one amino acid and the amino terminus of another.

The term “amino acid” as used herein refers to a naturally occurring or unnatural alpha or beta amino acid, wherein such natural or unnatural amino acids may be optionally substituted by one to four substituents, such as halo, for example F, Br, Cl or I or CF₃, alkyl, alkoxy, aryl, aryloxy, aryl(aryl) or diaryl, arylalkyl, arylalkyloxy, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, cycloalkylalkyloxy, optionally substituted amino, hydroxy, hydroxyalkyl, acyl, alkanoyl, heteroaryl, heteroaryloxy, cycloheteroalkyl, arylheteroaryl, arylalkoxycarbonyl, heteroarylalkyl, heteroarylalkoxy, aryloxyalkyl, aryloxyaryl, alkylamido, alkanoylamino, arylcarbonylamino, nitro, cyano, thiol, haloalkyl, trihaloalkyl and/or alkylthio.

The term “modified” as used herein refers to a composition that has been operably changed from one or more predominant forms found naturally to an altered form by any of a variety of methods, including genetic alteration or chemical substitution or degradation and comprising addition, subtraction, or alteration of biological components or substituents such as amino acid or nucleic acid residues, as well as the addition, subtraction or modification of protein post-translational modifications such as, without limitation, glycan, lipid, phosphate, sulfate, methyl, acetyl, ADP-ribosyl, ubiquitinyl, sumoyl, neddoyl, hydroxyl, carboxyl, amino, or formyl. “Modified” also comprises alteration by chemical or enzymatic substitution or degradation to add, subtract, or alter chemical moieties to provide a form not found in the composition as it exists in its natural abundance comprising a proportion of greater than 10%, or greater than 1%, or greater than 0.1%. The term “modified” is not intended to refer to a composition that has been altered incidentally as a consequence of manufacturing, purification, storage, or expression in a novel host and for which such alteration does not operably change the character of the composition.

The terms “fusion protein,” “protoxin fusion,” “toxin fusion,” “protoxin activator fusion” “protoxin proactivator fusion,” or “proactivator activator fusion” as used herein refer to a protein that has a peptide component operably linked to at least one additional component and that differs from a natural protein in the composition and/or organization of its domains. The additional component can be peptide or non-peptide in nature. Additional peptide components can be derived by natural production or by chemical synthesis, and in the case of a peptide component that acts as an inhibitor moiety, a cell-targeting moiety, or a cleavage site, the additional peptide components need not be based on any natural template but may be selected for the desired purpose from an artificial scaffold or random sequence or by diversification of an existing template such that substantially all of the primary sequence similarity is lost but the functional attributes are preserved. Non-peptide additional components can include one or more functional chemical species. The chemical species may comprise a linker or a cleavage site, each optionally substituted with one or more linkers that may provide flexible attachment of the chemical species to a polypeptide or to another chemical species.

The terms “operably linked” or “operable linkage” encompass the joining of two or more peptide components covalently or noncovalently or both covalently and noncovalently as well as the joining of one or more peptide components with one or more chemical species covalently or noncovalently or both covalently and noncovalently, as well as the joining of two or more chemical species covalently. Among suitable form of covalent linkage for peptide components are direct translational fusion, in which a single polypeptide is formed upon translation of mRNA, or post-translational fusion, achieved by operable linkage through chemical or enzymatic means or by operable linkage through natural intermolecular reactions such as the formation of disulfide bonds. Operable linkage may be performed through chemical or enzymatic activation of various portions of a donor molecule to result in the attachment of the activated donor molecule to a recipient molecule. Following operable linkage two moieties may have additional linker species between them, or no additional species, or may have undergone covalent joining that results in the loss of atoms from one or more moieties, for example as may occur following enzymatically induced operable linkage.

The term “transglutaminase” refers to a protein that catalyzes the formation of a covalent bond between a free amine group (e.g., protein- or peptide-bound lysine, or substituted aminoalkane such as a substituted cadaverine) and the gamma-carboxamide group of protein- or peptide bound glutamine. Examples of this family of proteins are transglutaminases of many different origins, including thrombin, factor XIII, and tissue transglutaminase from human and animals. A preferred embodiment comprises the use of a microbial transglutaminase (Yokoyama et al., Appl. Microbiol. Biotechnol. 64(4):447-454 (2004)) to catalyze an acyl transfer reaction between a first moiety containing a glutamine residue (acyl donor), located within a preferred sequence such as LLQG (SEQ ID NO:1), and a second moiety containing a primary amine group (acyl acceptor). It is preferable that the reactive glutamine residue is solvent exposed and located in an unstructured, i.e. flexible, segment of the polypeptide.

The term “sortase” refers to a protein from gram-positive bacteria that can recognize a conserved carboxylic sorting motif and catalyze a transpeptidation reaction to anchor surface proteins to the cell wall envelope (Dramsi et al., Res. Microbiol. 156(3):289-297 (2005)). A preferred embodiment comprises the use of Staphylococcus aureus sortase A or B to catalyze a transpeptidation reaction between a first moiety that is tagged with LPXTG (SEQ ID NO:2) or NPQTN (SEQ ID NO:3) at or near C-terminus, respectively for sortase A and sortase B, and a second moiety containing the dipeptide GG or GK at the N-terminus, or a primary amine group.

The term “immobilized sortase” refers to purified and active sortase enzyme that has been absorbed covalently or non-covalently to a solid support such as agarose. The enzyme can be chemically or enzymatically immobilized as described herein to matrices bearing a chemical functional group such as a free sulfhydril or amine. Alternatively, the enzyme can be modified and then immobilized through some specific interaction. For example, the sortase enzyme could be biotinylated and then immobilized via an indirect interaction with immobilized streptavidin.

The term “intein” refers to a protein that undergoes autoreaction resulting in the formation of novel peptide or amide linkages. Intein-mediated ligation is a well established method to perform protein-protein conjugation (Xu and Evans Methods 24(3):257-277 (2001)) as well as protein-small molecule conjugation (Wood, et al., Bioconjug. Chem. 15(2):366-372 (2004)). A self-splicing intein may be added to the C-terminus of a protein to be conjugated, and treated with a conjugation partner that contains cysteine that can undergo acyl transfer followed by S—N acyl shift to provide a stable amide linkage.

The term “toxin” or “protoxin” as used herein refers to a protein comprising one or more moieties that have the latent (protoxin) or manifest (toxin) ability to inhibit cell growth (cytostasis) or to cause cell death (cytotoxicity). Examples of such toxins or protoxins include, without limitation, Diphtheria toxin, Pseudomonas exotoxin A, Shiga toxin, and Shiga-like toxin, anthrax lethal factor toxin, anthrax edema factor toxin, pore-forming toxins or protoxins such as Proaerolysin, hemolysins, pneumolysin, Cryl toxins, Vibrio pro-cytolysin, or listeriolysin; Cholera toxin, Clostridium septicum alpha-toxin, Clostridial neurotoxins including tetanus toxin and botulinum toxin; gelonin; nucleic acid modifying agents such as ribonuclease A, human pancreatic ribonuclease, angiogenin, and pierisin-1, apoptosis-inducing enzymes such as caspases, and ribosome-inactivating proteins (RIPs) such as Ricin, Abrin, and Modeccin. A protoxin is a toxin precursor that must undergo modification to become an active toxin. Preferable forms of protoxins for the present invention include those that can be activated by a protoxin activator.

The term “selectively modifiable activation moiety” refers to an unnatural or not naturally found moiety of a protoxin or protoxin activator that, upon modification, converts a protoxin to a toxin or natively activatable protoxin or activates a protoxin proactivator or modifies the protoxin proactivator so that it becomes natively activatable. When the selectively modifiable activation moiety is a component of the protoxin fusion protein, modification of the modifiable activation moiety by the protoxin activator can result directly in the protoxin becoming toxic to the target cell, or can result in the protoxin assuming a form that is natively activatable to become toxic to the target cell. When the selectively modifiable activation moiety is a component of the protoxin proactivator protein, modification of the modifiable activation moiety by the proactivator activator can result directly in the proactivator becoming activated to a form that can modify the protoxin, or can result in the proactivator assuming a form that is natively activatable to become a form that can modify the protoxin. Natively activatable protoxins or proactivators comprise, for example, modification of the modifiable activation moiety such that it is sensitive to endogenous components of the target cell, or the environment surrounding the target cells. (e.g., a target cell specific protease or a ubiquitous protease).

The term “cell targeting moiety” as used herein refers to one or more protein domains that can bind to one or more cell surface targets, and thus can direct protoxins, protoxin activators, protoxin proactivators or proactivator activators to those cells. Such cell targeting moieties include, among others, antibodies or antibody-like molecules such as monoclonal antibodies, polyclonal antibodies, antibody fragments, single antibody domains and related molecules, such as scFv, diabodies, engineered lipocalins, camelbodies, nanobodies and related structures. Also included are soluble mediators, cytokines, growth factors, soluble receptor fragments, matrix fragments, or other structures that are known to have cognate binding structures on the targeted cell. In addition, protein domains that have been selected by diversification of an invariant or polymorphic scaffold, for example, in the formation of binding principles from fibronectin, anticalins, titin and other structures, are also included. Cell targeting moieties can also include combinations of moieties (e.g., an scFv with a cytokine and an scFv with a second scFv).

The term “artificially diversified polypeptide binder” as used herein refers to a peptide or polypeptide comprising at least one domain that has been made to comprise multiple embodiments as a result of natural or synthetic mutation, including addition, deletion and substitution, so as to provide an ensemble of peptides or polypeptides from which a high affinity variant capable of binding to the desired cell surface target can be isolated. Such artificially diversified binders can comprise peptides, for example as selected by phage display, ribosome display, RNA display, yeast display, cell surface display or related methods, or polypeptides, similarly selected, and typically diversified in flexible loops of robust scaffolds so as to provide antibody variable region mimetics or related binding molecules.

The term “cell surface target” as used herein refers to any structure operably exposed on the surface of a cell, including transient exposure as for example may be the consequence of fusion of intracellular vesicles with the plasma membrane, and that can be specifically recognized by a cell targeting moiety. A cell surface target may include one or more optionally substituted polypeptide, carbohydrate, nucleic acid, sterol or lipid moieties, or combinations thereof, as well as modifications of polypeptides, carbohydrate, nucleic acid, sterol or lipid moieties separately or in combination. A cell surface target may comprise a combination of optionally substituted polypeptide and optionally substituted carbohydrate, an optionally substituted carbohydrate and optionally substituted lipid or other structures operably recognized by a cell-targeting moiety. A cell surface target may comprise one or more such optionally substituted polypeptides, carbohydrates, nucleic acid, sterol or lipids in complexes, for example heteromultimeric proteins, glycan-substituted heteromultimeric proteins, or other complexes, such as the complex of a peptide with a major histocompatibility complex antigen. A cell surface target may exist in a form operably linked to the target cell through another binding intermediary. A cell surface target may be created by some intervention to modify particular cells with an optionally substituted small molecule, polypeptide, carbohydrate, nucleic acid, sterol or lipid. For example a cell surface target may be created by the administration of a species that binds to a cell of interest and thereby affords a binding surface for any of the protoxins, protoxin activators, protoxin proactivators or proactivator activators of the present invention.

The term “targeted cell” or “target cell” is used herein to refer to any cell that expresses at least two cell surface targets, which are the intended targets of one or more protoxins or protoxin activators or protoxin proactivators or proactivator activators.

The phrase “non toxic to a target cell” is used herein to refer to compositions that, when contacted with a target cell (i.e., the target cell to which the cell-targeting moiety of the protoxin activator is directed) under the conditions of use described in the present invention, do not significantly destroy or inhibit the growth of a target cell, that is do not reduce the proportion of viable cells in a target population, or the proportion of dividing cells in a target population, or the total proportion of cells in a target population by more than 50%, or 10%, or 1% or 0.1% under the preferred conditions of use. This phrase does not include fusion proteins that, when administered to a subject or contacted with a target cell, become activated by an endogenous factor, rendering them toxic to a target cell. By “target population” is meant cells that express targets for the cell targeting moieties of the present invention.

The term “natively activatable” as used herein refers to a composition or state that can be converted from an inactive form to an active form by the action of natural factors or environmental variables on, in, or in the vicinity of a target cell. In one embodiment “natively activatable” refers to toxins or protoxin activators that, either as a consequence of modification on a modifiable activation moiety, or not, have the property of being converted from an inactive form to an active form as a result of natural factors on, in, or in the vicinity of a target cell. In one embodiment, the natively activatable protein possesses a cleavage site for a ubiquitously distributed protease such as a furin/kexin protease. In another embodiment, the natively activatable protein possesses a cleavage site for a target cell-specific protease, such as a tumor-enriched protease. In yet another embodiment, the natively activatable protein can be activated by low pH in, on, or in the vicinity of, a target cell. In another embodiment, the natively activatable protein possesses a post-translational modification that is removable by an enzyme found in, on, or in the vicinity of a target cell. In another embodiment the natively activatable protein posesses a modifiable activation moiety that can be modified by an enzyme found in, on, or in the vicinity of a target cell. Examples of such non-protease enzymes include phosphatases, nucleases, and glycohydrolases.

The phrase “substantially promote” as used herein means to improve the referenced action or activity by 50%, or by 100%, or by 300%, or by 700% or more.

The term “natively targetable toxin” as used herein refers to a toxins that possess native cell-targeting moieties that permit the toxin to bind to cell surface targets.

The term “bacterial toxin” refers to a toxin that is selected from a repertoire that comprises at least 339 members including natural variants, serotypes, isoforms, and allelic forms, of which at least 160 are from Gram-positive bacteria and 179 are from Gram-negative bacteria. Most are extracellular or cell-associated and the rest are intracellular. Many bacterial toxins are enzymes, including ADP-ribosyltransferases, phospholipases, adenylate cyclases, metalloproteases, RNA N-glycosidase, glucosyl transferases, deamidases, proteases, and deoxyribonucleases (Alouf and Popoff, Eds. “The Comprehensive Sourcebook of Bacterial Protein Toxins, 3^(rd) Ed.” Academic Press. 2006).

The term “intracellular bacterial toxin” refers to bacterial toxins that enter cells through various trafficking pathways and act on targets in the intracellular compartment of eukaryotic cells.

The term “activatable AB toxin” as used herein refers to any protein that comprises a cell-targeting and translocation domain (B domain) as well as a biologically active domain (A domain) and that requires the action of an endogenous target cell protease on an activation sequence to substantially promote their toxic effect. AB toxins have the capability to intoxicate target cells without requirement for accessory proteins or protein-delivery structures such as the type III secretion system of gram negative bacteria. AB toxins typically contain a site that is sensitive to the action of ubiquitous furin/kexin-like proteases, and must undergo cleavage to become activated. According to the present invention, the term “activatable AB toxin” is meant to include modified AB toxins in which the endogenous cell-targeting domain is replaced by one or more heterologous cell-targeting moiety or in which one or more heterologous cell-targeting moiety is added to an intact endogenous cell-targeting domain, and the activation sequence is replaced with a modifiable activation moiety that may be modified by an exogenous activator.

The term “ribosome-inactivating protein” or “RIP” as used herein refers to enzymes that trigger the catalytic inactivation of ribosomes and other substrates. Such toxins are present in a large number of plants and have been found also in fungi, algae and bacteria. RIPs are currently classified as belonging to one of two types: type 1, comprising a single polypeptide chain with enzymatic activity, and type 2, comprising two distinct polypeptide chains, an. A chain equivalent to the polypeptide of a type 1 RIPs and a B chain with lectin activity. Type 2 RIPs known in the art may be represented by the formulae A-B, (A-B)₂, (A-B)₄ and or by polymeric forms comprising multiple B chains per A chain. Linkage of the A chain with B chain is through a disulfide bond. The toxic activity of RIPs is due to translational inhibition, a consequence of the hydrolysis of an N-glycosidic bond of a specific adenine base in a highly conserved loop region of the 28 S rRNA of the eukaryotic ribosome (Girbes et al, Mini Rev. Med. Chem. 4(5):461-76 (2004)).

The term “ADP-ribosylating toxin” refers to enzymes that transfer the ADP ribose moiety of β-NAD⁺ to a eukaryotic target protein. This process impairs essential functions of target cells, leading to cytostasis or cytotoxicity. Examples of bacterial ADP-ribosylating toxins include Diphtheria toxin, Pseudomonas aeruginosa exotoxin A, P. aeruginosa cytotoxic exotoxin S, pertussis toxin, cholera toxin, and heat-labile enterotoxins LT-I and LT-II from E. coil (Krueger and Barbieri, Clin. Microbiol. Rev. 8:34-47 (1995)). Examples of nonbacterial ADP-ribosylating toxins include the DNA ADP-ribosylating enzymes pierisin-1, pierisin-2, CARP-1 and the related toxins of the clams Ruditapes philippinarum and Corbicula japonica (Nakano et al. Proc Natl Acad Sci USA. 103(37):13652-7 (2006)). In addition, the application of in silico analyses have allowed the prediction of putative ADP-ribosylating toxins (Pallen et al. Trends Microbiol. 9:302-307 (2001).

ADP-ribosylating toxins of the present invention include those that can induce their own translocation across the target cell membranes, herein referred to as “autonomously acting ADP-ribosylating toxins,” which have no requirement for a type III secretion system or similar structure expressed by bacteria to convey the translocation of the toxin into the host cytoplasm by an injection pilus or related structure. Such autonomously acting ADP-ribosylating toxins can be modified with respect to their activation moiety and cell-targeting moiety and produced by methods well known in the art.

The term “dermonecrotic toxin” or “DNT” as used herein refers to virulence factors known as Bordetella dermonecrotic toxin and described in Bordetella species such as, without limitation, B. pertussis, B. parapertussis, or B. bronchoseptica.

The term “cytotoxic necrotizing factor” or “CNF” or “CNF1” or “CNF2” or “CNFY” as used herein refers to any virulence factor known as a cytotoxic necrotizing factor and described in gram-negative species such as, without limitation, Escherichia coli or Yersinia pseudotuberculosis.

The term “activatable ADP-ribosylating toxin” or “activatable ADPRT” as used herein refers to toxins that are functionally conserved enzymes produced by a variety of species that share the ability to transfer the ADP ribose moiety of β-NAD⁺ to a eukaryotic target protein and that require the action of an endogenous target cell protease on an activation sequence to substantially promote their toxic effect. This process impairs essential functions of target cells, leading to cytostasis or cytotoxicity. Examples of activatable bacterial ADPRTs are Diphtheria toxin, Pseudomonas aeruginosa exotoxin A, pertussis toxin, cholera toxin, and heat-labile enterotoxins LT-I and LT-II from E. coli (Krueger and Barbieri, Clin. Microbiol. Rev. 8:34-47 (1995); Holbourn et al. The FEBS J. 273:4579-4593(2006)). Examples of activatable nonbacterial ADP-ribosylating toxins include the DNA ADP-ribosylating enzymes from Cabbage butterfly, Pieris Rapae (Kanazawa et al Proc. Natl. Acad. Sci. 98:2226-2231 (2001)) and, by sequence homology, Pieris brassicae (Takamura-Enya et al., Biochem. Biophys. Res. Commun. 32:579-582 (2004)).

The term “activatable enzymatic toxin” refers to toxins that exert their toxic effect by enzymatic action and that require the action of an endogenous target cell protease on an activation sequence (e.g., a native or heterologous activation sequence) to substantially promote their toxic effect. The enzymatic action can be, for example and without limitation, an ADP-ribosyltransferase, a protease, a transglutaminase, a deamidase, a lipase, a phospholipase, a sphingomyelinase or a glycosyltransferase.

The term “pore-forming toxin” refers to toxins that create channels (pores) in the membrane of cells. The pore allows exchange of small molecules or ions between the extracellular and cytosolic space with an accompanying deleterious effect on the target cell incurred by such events as potassium efflux, sodium and calcium influx, the passage of essential small molecules through the membrane, cell lysis, or induced apoptosis. Some pore forming toxins are expressed as inactive toxins “protoxins” and become active only when modified in some manner at the cell surface while some pore-forming toxins require no modifications other than aggregation at the cell surface.

The term “activatable pore-forming toxins” refers to naturally occurring toxins that are expressed as inactive protoxins, and require an activation step in order for pore formation to occur. For example, many toxins require a furin cleavage event between a pro-domain and active pore-forming domain, essentially removing the pro-domain, in order for oligomerization and pore formation to occur.

Representative pore-forming toxins that require modification to become active include, Aeromonas hydrophila aerolysin, Clostridium perfringens ε-toxin, Clostridium septicum α-toxin, Escherichia coil prohaemolysin, hemolysins of Vibrio cholerae, and B. pertussis AC toxin (CyaA). The eukaryotic pore-forming protein, perforin, is inactive during the synthetic stage and activated by cleaving off a prodomain during maturation inside CTL and NK cells.

The term “reengineered activatable pore-forming toxin” or “RAPFT” refers to pore-forming toxins that have been modified to target only specific cell types in the context of combinatorial targeting. Typically, pore-forming agents are not specifically targeted towards diseased cells but act on healthy cells. Pore-forming agents often bind to common cellular markers such as carbohydrate groups, membrane proteins, glycosyl phosphatidylinositol anchors, and cholesterol. RAPFTs still retain the the cytolytic pore-forming activity, but the cell recognition and activation sites have been modified to specifically target cells possessing the targeted combination of surface markers.

The embodiments described herein comprise but are not limited to two types modifications. The first is a modification of the native cell-targeting portion of the toxin in order to target a specific class of cells using one or more optionally substituted cell-targeting moieties. The second modification introduces a modifiable activation moiety that can affect the pore-forming ability of the protoxin. When paired with a second targeting principle that can modify the modifiable activation moiety in a manner that activates the pore-forming toxin or converts it to a form that can be natively activated, the RAPFT can cause rapid loss of ion and small molecule gradients causing increased permeability, cytolysis, or apoptosis. These embodiments are unique with respect to previously reported pore-forming immunotoxins in that the activity that can convert the protoxin to the active toxin need not be endogenous to the target cell (Buckley, MacKenzie. 2006. Patent WO2007056867A1, Buckley. 2003. Patent WO03018611A2). An exogenous modifying moiety must be brought to the target cell via a second interaction between one or more cell-targeting moieties and one or more cell surface targets.

The term “translocation domain” of a toxin as used herein refers to an optional domain of a toxin (for example, a naturally occurring or modified toxin) that is necessary for translocation into the cytoplasm or a cytoplasm-contiguous compartment an active domain of a toxin. Prior to translocation the active domain may be located on the cell surface, or may have been conveyed from the cell surface into an intracellular space excluded from the cytoplasm, for example a vesicular compartment such as the endosome, lysosome, Golgi, or endoplasmic reticulum. Examples of such domains are the translocation domain of DT (residues 187-389) and the translocation domain of Pseudomonas exotoxin A (residues 253-364). Not all toxins contain translocation domains (e.g., pore forming toxins).

The term “Diphtheria toxin” or “DT” as used herein a protein selected from the family of protoxins, the prototype of which is a 535 amino acid polypeptide encoded by lysogenic bacteriophage of Corynebacterium diphtheriae. The prototypical diphtheria toxin contains three domains: a catalytic domain (residues 1-186), a translocation domain (residues 187-389), and a cell-targeting moiety (residues 390-535). The catalytic domain and the translocation domain are linked through a furin cleavage site (residues 190-195: RVRR↓SV (SEQ ID NO:4). Diphtheria toxin binds to a widely expressed growth factor expressed on the cell surface via its cell-targeting moiety and is internalized into the endosomal compartment of the cell, where furin cleaves at RVRR↓SV and the catalytic domain is translocated to the cytosol. In the cytosol, the catalytic domain catalyzes ADP-ribosylation of elongation factor 2 (EF-2), thereby inhibiting protein synthesis and inducing cytotoxicity or cytostasis.

The terms “modified DT,” or “engineered DT” are used interchangeably herein to describe a recombinant or synthetic DT that is modified to confer amino acid sequence changes as compared with that of any natural DT, including extending, shortening, and replacing amino acid sequences within the original sequence. In particular, the terms may refer to DT proteins with sequence changes at the furin cleavage site to provide a modifiable activation moiety that is a recognition site for proteases other than furin, and/or DT fusion proteins with their native cell-targeting moiety removed or changed to other cell-targeting ligands. The term may also refer to DT with modifications such as glycosylation and PEGylation.

The term “DT fusion” as used herein refers to a fusion protein containing a DT or modified DT, for example, and a polypeptide that can bind to a targeted cell surface. The DT or modified DT is preferably located at the N-terminus of the fusion protein and the cell-targeting polypeptide attached to the C-terminus of the DT or modified DT. When discussed in the context of fusion toxins, “modified DT” may simply be referred to as “DT.”

The term “Pseudomonas exotoxin A,” “PE” or “PEA” as used herein refers to a protein selected from the family of protoxins, the prototype of which is an ADP-ribosyltransferase produced by Pseudomonas aeruginosa. The prototypical PEA is a 638 amino acid protein and has the following domain organization: an N-terminus receptor binding moiety (residues 1-252), a translocation domain (residues 253-364) and a C-terminal catalytic domain (residues 405-613). PEA is internalized into eukaryotic cells via receptor-mediated endocytosis and transported to ER, where it was cleaved at the furin cleavage site (residues 276-281: RQPR↓GW (SEQ ID NO:5)). The catalytic domain is translocated into the cytosol, where it catalyzes ADP-ribosylation of EF2, resulting in cell killing.

The term “modified PEA” or “engineered PEA” are used interchangeably herein to describe a recombinant or synthetic PEA protein that is modified to confer amino acid sequence changes compared with that of natural PEA, including extending, shortening, and replacing amino acid sequences within the original sequence, addition of linkers, of modifiable activation moieties or cell-targeting moieties. In particular, the terms may refer to PEA proteins with sequence changes at the furin cleavage site to provide a modifiable activation moiety that is capable of being modified by a protoxin activator, and/or PEA fusion proteins with their native cell-targeting moieties removed or changed to therapeutically desirable cell-targeting moieties. The term may also refer to PEA with amino acid covalent modifications or containing unnatural amino acids and or variants derived by optional substitution with other moieties such as to induce glycosylation and/or PEGylation. The term may also refer to PEA with alterations to the C terminus to increase specificity or activity, for example to the C-terminal endoplasmic reticulum retention sequence, more specifically to consensus versions of such sequence and variants.

The term “PEA fusion” as used herein refers to a fusion protein containing a PEA or modified PEA, for example, and a cell-targeting moiety that can bind to a targeted cell surface. The PEA or modified PEA is preferably located at the C-terminus of the fusion protein and the cell-targeting moiety is preferably attached to the N-terminus of the PEA or modified PE. When discussed in the context of fusion toxins, “modified PEA” may simply be referred to as “PEA”.

The term “Vibrio Cholerae exotoxin A” or “VCE” as used herein refers to a protein selected from the family of protoxins, the prototype of which is a diphthamide-specific toxin encoded by the toxA gene of Vibrio cholerae. The prototypical VCE possesses a conserved DT-like ADP-ribosylation domain, and adopts an overall domain structure very similar to that of Pseudomonas exotoxin A (PEA), with moderate amino acid sequence identity (˜33%). Like PEA, the VCE possesses an N-terminal cell-targeting moiety, followed by a translocation domain and a C-terminal ADP-ribosyltransferase. A putative furin cleavage site (RKPK↓DL (SEQ ID NO:6)) is located near the N-terminus of the putative translocation domain.

The term “modified VCE”, “modified VCE”, or “engineered VCE” are used interchangeably herein to describe a recombinant or synthetic VCE protein that is modified to confer amino acid sequence changes as compared with that of VCE, including extending, shortening, and replacing amino acid sequences within the original sequence. In particular, the terms may refer to VCE proteins with sequence changes at the furin cleavage site to provide a mutated sequence that is a recognition site for proteases other than furin, and/or VCE fusion proteins with their native cell-targeting moiety removed or changed to cell-targeting ligands. The term may also refer to VCE with amino acid covalent modifications such as glycosylation and PEGylation.

The term “VCE fusion” as used herein refers to a fusion protein containing a VCE or modified VCE, for example, and a polypeptide that can bind to a targeted cell surface. The VCE or modified VCE is preferably located at the C-terminus of the fusion protein and the cell-targeting polypeptide attached to the N-terminus of the VCE or modified VCE. When discussed in the context of fusion toxins, “modified VCE” may simply be referred to as “VCE.”

The terms “proaerolysin” or “aerolysin” as used herein refers a protein selected from the family of bacterial pore forming toxin encoded by Aeromonas species, the prototype of which is a pore-forming toxin from Aeromonas hydrophila. The prototypical proaerolysin is composed of four domains: N-terminus Domain 1 (residues 1-82) that can bind to N-linked glycan of its glycosylated GPI-anchored receptors, Domain 2 (residues 83-178 & 311-398) that binds to the glycan core of the GPI-anchor, and non-contiguous Domains 3 and 4 (residues 179-470) that are involved in heptamerization and pore formation. Located at the C-terminus of Domain 4 is a propeptide that is sensitive to furin cleavage at its recognition sequence just upstream (residues 427-432 KVRR↓AR (SEQ ID NO:7)). Furin removal of the propeptide promotes formation of a ring-like heptamer structure, which insert into a lipid membrane to form a pore and cause cell death. Domain I is also known as the small lobe, and Domains 2, 3, and 4 as a whole are known as the large lobe.

The terms “modified aerolysin”, or “engineered aerolysin” are used interchangeably herein to describe a recombinant or synthetic aerolysin protein that is modified to confer amino acid sequence changes as compared with that of aerolysin, including extending, shortening, and replacing amino acid sequences within the original sequence. In particular, the terms may refer to aerolysin proteins with sequence changes at the furin cleavage site to provide a mutated sequence that is a recognition site for proteases other than furin, and/or aerolysin fusion proteins with the native cell-targeting moiety 1 (small lobe) removed or changed to cell-targeting ligands. The term may also refer to aerolysin with amino acid covalent modifications such as glycosylation and PEGylation. The term may also refer to functional fragments of aerolysin.

The term “aerolysin fusion” as used herein refers to a fusion protein containing an aerolysin or modified aerolysin, for example, and a polypeptide that can bind to a targeted cell surface. The aerolysin or modified aerolysin is preferably located at the C-terminus of the fusion protein and the cell-targeting polypeptide attached to the N-terminus of the aerolysin or modified aerolysin. When discussed in the context of fusion toxins, “modified aerolysin” may simply be referred to as “aerolysin.”

The term “protoxin activator” is meant to include a protein that modifies a protoxin such that the toxin becomes able to inhibit cell growth or to cause cell death.

The term “modification domain” as used herein refers to a polypeptide that selectively modifies a selectively modifiable activation domain on a target molecule. Such modification is meant to include modification of the polypeptide structure of the target molecule or the addition or removal of a chemical moiety. Examples of modification domains are polypeptides that contain protease activity, phosphatase activity, kinase activity, and other modifications as described herein.

The term “enzyme” as used herein refers to a catalyst that mediates a specific chemical modification (i.e., the addition, removal, or substitution of a chemical component) of a “substrate”. The term enzyme is meant to include proteases, phophatases, kinases, or other chemical modifications as described herein.

The term “substrate” as used herein refers to the specific molecule, or portion of a molecules, that is recognized and chemically modified by a particular enzyme.

The term “protease” as used herein refers to compositions that possess proteolytic activity, and preferably those that can recognize and cleave certain peptide sequences specifically. In one particular embodiment, the specific recognition site is equal to or longer than that of the native furin cleavage sequence of four amino acids, thus providing activation stringency comparable to, or greater than, that of native toxins. A protease may be a native, engineered, or synthetic molecule having the desired proteolytic activity. Proteolytic specificity can be enhanced by genetic mutation, in vitro modification, or addition or subtraction of binding moieties that control activity.

The term “heterologous” as used herein refers to a composition or state that is not native or naturally found, for example, that may be achieved by replacing an existing natural composition or state with one that is derived from another source. Thus replacement of a naturally existing, for example, furin-sensitive, cleavage site with the cleavage site for another enzyme, constitutes the replacement of the native site with a heterologous site. Similarly the expression of a protein in an organism other than the organism in which that protein is naturally expressed constitutes a heterologous expression system and a heterologous protein.

The term “exogenous” as used herein refers to any protein that is not operably present in, on, or in the vicinity of, a targeted host cell. By operably present it is meant that the protein, if present, is not present in a form that allows it to act in the way that the therapeutically supplied protein is capable of acting. Examples of protoxin-activating moiety that may be present but not operably present include, for example, intracellular proteases, phosphatases or ubiquitin C-terminal hydrolases, which are not operably present because they are in a different compartment than the therapeutically supplied protease, phosphatase or ubiquitin C-terminal hydrolase (which when therapeutically supplied is either present on the surface of the cell or in a vesicular compartment topologically equivalent to the exterior of the cell) and cannot act on the protoxin in a way that would cause its activation. A protein may also be present but not operably present if it is found in such low quantities as not to significantly affect the rate of activation of the protoxin or protoxin proactivator, for example to provide a form not operably found in, on, or in the vicinity of, a targeted cell in a proportion of greater than 10%, or greater than 1%, or greater than 0.1% of the proportion that can be achieved by exogenous supply of a minimum therapeutically effective dose. As a further non-limiting example, replacement of a furin-sensitive site in a therapeutic protein with a site for a protease naturally found operably present on, in, or In the vicinity of a targeted host cell constitutes a heterologous replacement that can be acted on by an endogenous protease. Replacement of a furin-sensitive site in a therapeutic protein with a site for a protease not naturally found operably present in the vicinity of a targeted host cell constitutes a heterologous replacement that can be acted on by an exogenous protease.

The term “PEGylation” refers to covalent or noncovalent modifications of proteins with polyethylene glycol polymers of various sizes and geometries, such as linear, branched and dendrimer and may refer to block copolymers incorporating polyethylene glycol polymers or modified polymers with additional functionality, such as may be useful for the therapeutic action of a modified toxin. For example a polyethylene glycol moiety may join a modifiable activation sequence to an optional inhibitor sequence or may join one or more cell-targeting moieties to a modified toxin. Many strategies for PEGylating proteins in a manner that is consistent with retention of activity of the conjugated protein have been described in the art. These include conjugation to a free thiol such as a cysteine by alkylation or Michael addition, attachment to the N-terminus by acylation or reductive alkylation, attachment to the side chain amino groups of lysine residues, attachment to glutamine residues using transglutaminase, attachment to the N-terminus by native ligation or Staudinger ligation, or attachment to endogenous glycans, such as N-linked glycans or O-liked glycans. Numerous glycan addition strategies are known, including hydrazone formation with aldehydes generated by periodate oxidation, Staudinger ligation with glycan azides incorporated by metabolic labeling, and glycan substitution technology. Examples of noncovalent modification include the reaction of a high affinity ligand-substituted PEG with a protein domain binding such ligand, as for example the reaction of a biotin-substituted PEG moiety with a streptavidin or avidin fusion protein.

The term “PEG” refers to an optionally substituted polyethylene glycol moiety that may exist in various sizes and geometries, such as linear, branched or dendrimer and may refer to block copolymers or modified polymers with additional functionality, such as may be useful for the therapeutic action of a modified toxin. The number of optionally substituted or unsubstituted ethylene glycol moieties in a PEG moiety is at least two.

The term “PEGylated” refers to a composition that has undergone reversible or irreversible attachment of a PEG moiety.

The term “thiol-specific PEGylation” refers to attachment of an optionally substituted thiol-reactive PEG moiety to one or more thiol groups of a protein or protein substituent. The target of thiol-directed PEGylation can be a cysteine residue, or a thiol group introduced by chemical reaction, such as by the reaction of iminothiolane with lysine epsilon amino groups or N-terminal alpha amino or imino groups. A number of highly specific chemistries have been developed for thiol-directed PEGylation, i.e., PEG-ortho-pyridyl-disulfide, PEG-maleimide, PEG-vinylsulfone, and PEG-iodoacetamide. In addition to the type of thiol specific conjugation chemistry, commercially available thiol-reactive PEGs also vary in terms of size, linear or branched, and different end groups including hydroxyl, carboxylic acid, methoxy, or other alkoxy groups.

The term “carboxyl-reactive PEGylation” refers to the reaction of a protein or protein substituent with an optionally substituted PEG moiety capable of reacting with a carboxyl group, such as a glutamate or aspartate side chain or the C-terminus of a protein. The carboxyl groups of a protein can be subjected to carboxyl-reactive PEGylation using PEG-hydrazide when the carboxyl groups are activated by coupling agents such as N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) at acidic pH.

The term “amine-reactive PEGylation” refers to the reaction of a protein or protein substituent with an optionally substituted PEG moiety capable of reacting with an amine, such as a primary amine or a secondary amine. A common route for amine-reactive PEGylation of proteins is to use a PEG containing a functional group that reacts with lysines and/or an N-terminal amino or imino group (Roberts et al. Adv. Drug Deliv. Rev. 54(4):459-476 (2002)). Examples of amine-reactive PEGs include PEG dichlorotriazine, PEG tresylate, PEG succinimidyl carbonate, PEG benzotriazole carbonate, PEG p-nitrophenyl carbonate, PEG carbonylimidazole, PEG succinimidyl succinate, PEG propionaldehyde, PEG acetaldehyde, and PEG N-hydroxysuccinimide.

The term “N-terminal PEGylation” refers to attachment of an optionally substituted PEG moiety to the amino terminus of a protein. Preferred protein fusions or protein hybrids for N-terminal PEGylation have at least one N-terminal amino group. N-terminal PEGylation can be carried out by reaction of an amine-reactive PEG with a protein, or by reaction of a thioester-terminated PEG with an N-terminal cysteine in the reaction known as native chemical ligation, or by reaction of a hydrazide, hydrazine or hydroxylamine terminated PEG with an N terminal aldehyde formed by periodate oxidation of an N-terminal serine or threonine residue. Preferably, a PEG-protein conjugate contains 1-5 PEG substituents, and may be optimized experimentally. Multiple attachments may occur if the protein is exposed to PEGylation reagents in excess. Reaction conditions, including protein:PEG ratio, pH, and incubation time and temperature may be adjusted to limit the number and/or sites of the attachments. Modification at active site(s) within a fusion protein may be prevented by conducting PEGylation in the presence of a substrate, reversible inhibitor, or a binding protein. A fusion protein with the desired number of PEG substitutions may also be obtained by isolation from a more complex PEGylated fusion protein mixture using column chromatography fractionation.

The term “unnatural amino acid-reactive PEGylation” refers to the reaction of a protein or protein substituent with an optionally substituted PEG moiety capable of reacting with unnatural amino acids bearing reactive functional groups that may be introduced into a protein at certain sites utilizing modified tRNAs. In particular, para-azidophenylalanine and azidohomoalanine may be specifically incorporated into proteins by expression in yeast (Deiters et al. Bioorg. Med. Chem. Lett. 14(23):5743-5 (2004)) and in E. coli (Kiick et al. Proc. Natl. Acad. Sci. USA. 99(1):19-24 (2002)), respectively. These azide modified residues can selectively react with an alkyne derivatized PEG reagent to allow site specific PEGylation.

The term “glycan-reactive PEGylation” refers to the reaction of a protein or protein substituent with an optionally substituted PEG moiety capable of reacting with a glycosylated protein and the proteins containing N-terminus serine or threonine may be PEGylated followed by selective oxidation. Carbohydrate side chains may be oxidized enzymatically, or chemically using sodium periodate to generate reactive aldehyde groups. N-terminus serine or threonine may similarly undergo periodate oxidation to afford a glyoxylyl derivative. Both aldehyde and glyoxylyl groups can selectively react with PEG-hydrazine or PEG-amine.

The term “enzyme-catalyzed PEGylation” refers to the reaction of a protein or protein substituent with an optionally substituted PEG moiety through one or more enzyme catalyzed reactions. One such approach is to use transglutaminases, a family of proteins that catalyze the formation of a covalent bond between a free amine group and the gamma-carboxamide group of protein- or peptide-bound glutamine. Examples of this family of proteins include transglutaminases of many different origins, including thrombin, factor XIII, and tissue transglutaminase from human and animals. A preferred embodiment comprises the use of a microbial transglutaminase, to catalyze a conjugation reaction between a protein substrate containing a glutamine residue embedded within a peptide sequence of LLQG (SEQ ID NO:8) and a PEGylating reagent containing a primary amino group (Sato Adv. Drug Deliv. Rev. 54(4):487-504 (2002)). Another example is to use a sortase to induce the same conjugation. Accordingly a substituted PEG moiety is provided that is endowed with LPXTG (SEQ ID NO:2) or NPQTN (SEQ ID NO:3), respectively for sortase A and sortase B, and a second moiety such as a polypeptide containing the dipeptide GG or GK at the N-terminus, or a primary amine group, or the dipeptide GG or GK attached to a linker, and said sortase A or sortase B is then provided to accomplish the joining of the PEG moiety to the second moiety. Alternatively, said LPXTG (SEQ ID NO:2) or NPQTN (SEQ ID NO:3) can be provided at the C-terminus of a polypeptide to be modified and the PEG moiety can be supplied that is substituted with a GG or GK or a primary amine, and the sortase reaction performed.

The term “glycoPEGylation” refers to the reaction of a protein with an optionally substituted PEG moiety through enzymatic GalNAc glycosylation at specific serine and threonine residues in proteins expressed in a prokaryotic host, followed by enzymatic transfer of sialic acid conjugated PEG to the introduced GalNAc (Defrees et al. Glycobiology. 16(9):833-843 (2006)).

The term “intein-mediated PEGylation” refers to the reaction of a protein with an optionally substituted PEG moiety through an intein domain that may be attached to the C-terminus of the protein to be PEGylated, and is subsequently treated with a cysteine terminated PEG to afford PEGylated protein. Such intein-mediated protein conjugation reactions are promoted by the addition of thiophenol or triarboxylethylphosphine (Wood, et al., Bioconjug. Chem. 15(2):366-372 (2004)).

The term “reversible PEGylation” refers to the reaction of a protein or protein substituent with an optionally substituted PEG moiety through a linker that can be cleaved or eliminated, liberating the PEG moiety. Preferable forms of reversible PEGylation involve the use of linkers that are susceptible to various activities present at the cell surface or in intracellular compartments, and allow the useful prolongation of plasma half-life and/or reduction of immunogenicity while still permitting the internalized or cell-surface-bound protoxin or protoxin proactivator or proactivator activator to carry out their desired action without inhibition or impediment by the PEG substitution. Examples of reversible PEGylation linkers include linkers susceptible to the action of cathepsins, furin/kexin proteases, and lysosomal hydrolases such as neuraminidases, nucleases and glycol hydrolases.

The term “administering” and “co-administering” as used herein refer to the application of two or more fusion proteins, simultaneously and/or sequentially to an organism in need of treatment. The sequential order, time interval, and relative quantity of the application may be varied to achieve an optimized selective cytotoxic or cytostatic effect. It may be preferable to use one agent in large excess, or to use two agents in similar quantities. One agent may be applied significantly before the addition of the second agent, or they may be applied in closer intervals or at the same time. In addition administering and co-administering may include injection or delivery from more than one site, for example by injection into two different anatomical locations or by delivery by more than one modality, such as by aerosol and intravenous injection, or by intravenous and intramuscular injection.

The term “selective killing” is used herein to refer to the killing, destroying, or inhibiting of more cells of one particular population than another, e.g., by a margin of 99:1 or above, 95:5 or above, 90:10 or above, 85:15 or above, 80:20 or above, 75:25 or above, 70:30 or above, 65:35 or above, or 60:40 or above.

The term “destroying or inhibiting a target cell” is used herein to refer to reducing the rate of cellular division (cytostasis) or causing cell death (cytotoxicity) of a particular cell type (e.g., a cell expressing the desired cell surface targets). Cytostasis or cytotoxicity may be achieved, for example, by the induction of differentiation of the cell, apoptosis of the cell, death by necrosis of the cell, or impairment of the processes of cellular division.

The term “glycosylation” refers to covalent modifications of proteins with carbohydrates. Glycosylation can be achieved through N-glycosylation or O-glycosylation. An introduction of consensus N-linked glycosylation sites may be preferred when the proteins are to be produced in a mammalian cell line or cell lines that create a glycosylation pattern that are innocuous to humans.

Human “granzyme B” (GrB) is a member of the granzyme family of serine proteases known to be involved in apoptosis. Specifically, GrB has been shown to cleave only a limited number of natural substrates, e.g., pro-caspase-3 and Bid. It has been shown that GrB is an enzyme with high substrate sequence specificity because of the requirement for interactions with an extended peptide sequence in the substrate for efficient catalysis, i.e., a consensus recognition sequence of IEPD (SEQ ID NO:9). GrB is a single chain and single domain serine protease and is synthesized in a pro-form, which is activated by removal of the two amino acid pro-peptide by dipeptidyl peptidase I (DPPI (SEQ ID NO:10). In the present invention, the term GrB for example refers to the mature form, i.e., the form without the propeptide.

Human “Granzyme M” (GrM) is another member of the granzyme family of serine proteases that is specifically found in granules of natural killer cells and is implicated in the induction of target cell death. It has been shown that GrM is an enzyme with high substrate sequence specificity because of the requirement for interactions with at least four amino acids in the peptide substrate for efficient catalysis, i.e., a preferred recognition sequence of KVPL (SEQ ID NO:11).

The term “potyviral protease” refers to any of a variety of proteases encoded by members of the plant virus family Potyviridae and exhibiting high cleavage specificity. “Potyviral protease” encompasses the natural proteases as well as engineered variants generated by genetic mutation or chemical modification. The term “tobacco etch virus protease” or “TEV protease” refers to natural or engineered variants of a 27 kDa cysteine protease exhibiting stringent sequence specificity. It is widely used in biotechnology for removal of affinity tags of recombinant proteins. TEV protease recognizes a seven amino acid recognition sequence EXXYXQ↓S/G (SEQ ID NO:12), where X is any residue.

The term “picornaviral protease” refers to any of a variety of proteases encoded by members of the animal virus family Picornaviridae and exhibiting high cleavage specificity. “picornaviral protease” encompasses the natural proteases as well as engineered variants generated by genetic mutation or chemical or enzymatic modification. The term “human Rhinovirus 3C consensus protease” refers to a synthetic picornaviral protease that is created by choice of a consensus sequence derived from multiple examples of specific rhinoviral proteases.

The term “retroviral protease” refers to any of a variety of proteases encoded by members of the virus family Retroviridae. “HIV protease” encompasses the natural proteases as well as engineered variants generated by genetic mutation or chemical or enzymatic modification.

The term “coronaviral protease” refers to any of a variety of proteases encoded by members of the animal virus family Coronaviridae and exhibiting high cleavage specificity. “coronaviral protease” encompasses the natural proteases as well as engineered variants generated by genetic mutation or chemical or enzymatic modification. The term “SARS protease” refers to a coronaviral protease encoded by any of the members of the family Coronaviridae inducing the human syndrome SARS.

By “substantially identical” is meant a nucleic acid or amino acid sequence that, when optimally aligned, for example using the methods described below, share at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a second nucleic acid or amino acid sequence, e.g., a SAA sequence. “Substantial identity” may be used to refer to various types and lengths of sequence, such as full-length sequence, epitopes or immunogenic peptides, functional domains, coding and/or regulatory sequences, exons, introns, promoters, and genomic sequences. Percent identity between two polypeptides or nucleic acid sequences is determined in various ways that are within the skill in the art, for instance, using publicly available computer software such as Smith Waterman Alignment (Smith, T. F. and M. S. Waterman (1981) J Mol Biol 147:195-7); “BestFit” (Smith and Waterman, Advances in Applied Mathematics, 482-489 (1981)) as incorporated into GeneMatcher Plus™, Schwarz and Dayhof (1979) Atlas of Protein Sequence and Structure, Dayhof, M. O., Ed pp 353-358; BLAST program (Basic Local Alignment Search Tool; (Altschul, S. F., W. Gish, et al. (1990) J Mol Biol 215: 403-10), BLAST-2, BLAST-P, BLAST-N, BLAST-X, WU-BLAST-2, ALIGN, ALIGN-2, CLUSTAL, or Megalign (DNASTAR) software. In addition, those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the length of the sequences being compared. In general, for proteins or nucleic acids, the length of comparison can be any length, up to and including full length (e.g., 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%). Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.

By the term “cancer cell” is meant a component of a cell population characterized by inappropriate accumulation in a tissue. This inappropriate accumulation may be the result of a genetic or epigenetic variation that occurs in one or more cells of the cell population. This genetic or epigenetic variation causes the cells of the cell population to grow faster, die slower, or differentiate slower than the surrounding, normal tissue. The term “cancer cell” as used herein also encompasses cells that support the growth or survival of a malignant cell. Such supporting cells may include fibroblasts, vascular or lymphatic endothelial cells, inflammatory cells or co-expanded nonneoplastic cells that favor the growth or survival of the malignant cell. The term “cancer cell” is meant to include cancers of hematopoietic, epithelial, endothelial, or solid tissue origin. The term “cancer cell” is also meant to include cancer stem cells. The cancer cells targeted by the fusion proteins of the invention include those set forth in Table 1.

A major limitation of all previously described approaches to targeting cells is their reliance on endogenous proteases, which may not be present on all tumors, or may be present in inadequate abundance, or may be shed in substantial quantities, leading to nonspecific activation of the toxin. The present invention differs from existing methods by its independence from endogenous tumor proteases. The combinatorial toxins of the present invention can be used on tumor cells or other undesired cells that have no appropriate endogenous protease activity.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a schematic depiction of expression cassettes for GrB-anti-CD19 and DT-anti-CD5 fusion proteins. GrB-anti-CD19 was produced from 293ETN cells as secreted protein and an N-terminal FLAG tag (N), which was removed by enterokinase to yield an enzymatically active fusion protein. Mature human Granzyme B and anti-CD19 ScFv are linked via a (G₄S)₃ linker (L). A polyhistidine tag (H) is added to the C-terminus of anti-CD19 ScFv for detection and purification. Expression of DT-anti-CD5 fusion protein is driven by the AOX1 promoter. The fusion protein is constructed in a form to be secreted into culture media by attachment of the yeast α factor signal peptide at the N-terminus (S). The α factor signal peptide is removed by protease Kex2 during the process of secretion. The endogenous furin cleavage site of the DT gene is replaced by a granzyme B cleavage site (IEPD↓SG (SEQ ID NO:13)) or an HRV 3C protease cleavage site (ALFQ↓GP (SEQ ID NO:14)). The toxin moiety and anti-CD5 ScFv are linked via a (G₄S₃) linker (L). A polyhistidine tag (H) is present at the C-terminus of anti-CD5 ScFv for detection and purification.

FIG. 1B is an electrophoretic gel showing cleavage of DT-anti-CD5 fusion protein by granzyme B proteolytic activity. Purified DT-anti-CD5 fusion protein with an additional N-terminal FLAG tag was incubated with either mouse granzyme B or purified GrB-anti-CD19 fusion protein at room temperature overnight. Reaction products were separated by 4-12% SDS-PAGE and immunoblotted with anti-FLAG antibody. Full length protein and cleaved products are indicated by arrows.

FIG. 1C is an electrophoretic gel showing cleavage of DT-anti-CD5 with a granzyme B site (lanes 1 to 4) or an HRV 3C protease site (lanes 5 to 8) with various proteases. Reactions were carried out at room temperature overnight. The products were detected with anti-His tag antibody. Full length protein and cleaved products are indicated by arrows. Asterisks in lanes 3 and 7 indicate unknown proteins present in the HRV 3C protease sample. G: granzyme B; 3C: HRV 3C protease; F: furin.

FIG. 2 shows generation of the reporter cell line. Cultured cells from sorted CD5 expressing Raji cells (CD5⁺Raji) were analyzed by cytometry for CD5 and CD19 expression. The Raji cells only express CD19, whereas CD5⁺Raji cells express both CD5 and CD19.

FIG. 3A is a graph showing GrB-anti-CD19 alone was not toxic to cells. The cells were incubated with GrB-anti-CD19 at the concentrations indicated below the graph. The relative cytotoxicity of the fusion proteins in comparison to buffer treated controls was determined by [³H]-leucine uptake.

FIG. 3B is a graph showing DT-anti-CD5 selectively kills CD5⁺Raji cells in the presence of GrB-anti-CD19. The cells were treated with 1.3 nM GrB-anti-CD19 and various concentrations of DT-anti-CD5. Nonlinear regression analysis was performed using the GraphPad Prism 4 program.

FIG. 4A and FIG. 4B are graphs showing cytotoxicity assays to determine the EC50 of GrB-anti-CD19 in the presence of fixed concentrations of DT-anti-CD5 (0.3 nM, 1.0 nM, and 3.0 nM) using non-target Raji cells (FIG. 4A) and target CD5⁺Raji cells (FIG. 4B). Nonlinear regression analysis was performed using the GraphPad Prism 4 program.

FIG. 5 is a graph showing cytotoxicity assays to determine the EC50 of DT-anti-CD5 in the presence of a fixed concentration of GrB-anti-CD19 (2 nM) using CD5⁺Raji cells. Nonlinear regression analysis was performed using the GraphPad Prism 4 program.

FIG. 6A and FIG. 6B are graphs showing that the combination of DT-anti-CD5 and GrB-anti-CD19 is selectively toxic to CD19⁺Jurkat cells. The relative cytotoxicity of the fusion protein(s) in comparison to buffer treated controls was determined by [³H]-leucine uptake. FIG. 6A, Jurkat or CD19⁺ Jurkat cells were incubated with 1.0 nM GrB-anti-CD19 and various concentrations of DT-anti-CD5 as shown in the graph. FIG. 6B, Jurkat or CD19⁺Jurkat cells were pre-treated with 1.0 nM GrB-anti-CD19 at 4° C. for 30 min. GrB-anti-CD19 was then washed away, replaced with a medium with or without 10 nM DT-anti-CD5, and incubated at 37° C. for 20 hours. For control experiments, cells were treated with 10 nM DT-anti-CD5±1.0 nM GrB-anti-CD19 and incubated at 37° C. for 20 hours.

FIG. 7A is a schematic depiction of anti-CD5-PE and DT-anti-CD5 fusion proteins. Artificially synthesized PE gene was fused with the anti-CD5 ScFv gene used in the construction of DT-anti-CD5. Several key features of anti-CD5-PE, including a granzyme B site that replaces the furin site of PE, a C-terminal 6 His tag (H), an N-terminal FLAG tag (N), and an ER retention signal (KDEL (SEQ ID NO:15)) are shown.

FIG. 7B and FIG. 7C are photographs showing 4-12% gradient SDS-PAGE analysis of purified anti-CD5-PE and proteolytic products after mouse GrB treatment, respectively. Anti-CD5-PE was expressed in E. coli and was purified from the inclusion body. After refolding, the protein was further purified by gel filtration (Sephadex 75) or by using M2 anti-FLAG tag antibody beads. The refolded anti-CD5-PE is incubated with mouse granzyme B digestion at 30° C. for 3 hours.

FIG. 8 is graph showing the use of anti-CD5-PE in the context of combinatorial targeting. Cytotoxicity assays were performed with 1.0 nM GrB-anti-CD19 and various concentrations of anti-CD5-PE using four different cell lines, among them CD5⁺Raji and CD5⁺JVM3 as target cell lines and Raji and JVM3 as non-target cell lines. Nonlinear regression data analysis was performed as described above. Selective killing of the target cell lines was observed.

FIG. 9A is a sequence alignment showing the sequence comparison of pseudomonas exotoxin A (PE) (SEQ ID NO:16) with a PE-like toxin from a Vibrio Cholerae environmental isolate (SEQ ID NO:17) TP using BLAST.

FIG. 9B is a table showing an analysis of overall sequence identity between PE and VCE as well as sequence identity of individual domains of PE and VCE.

FIG. 9C is a sequence alignment showing the sequence of the putative furin cleavage site in VCE (SEQ ID NO:18) in comparison with the furin cleavage sites of PE (SEQ ID NO:19) and DT (SEQ ID NO:20). Residues that are critical for efficient in vitro furin cleavage are highlighted in gray.

FIG. 10A is a schematic depiction of anti-CD5-VCE. For comparison, the structure of anti-CD5-PE is also shown.

FIG. 10B is a photograph showing a 4-12% SDS-PAGE analysis of purified anti-CD5-VCE and anti-CD5-PE visualized by Coomassie Blue staining. Expression, purification, and refolding of anti-CD5-VCE were carried out following the same protocol that produced functional anti-CD5-PE.

FIG. 11 is a graph showing cytotoxicity assay results of VCE-based combinatorial targeting agents using CD5⁺Raji cells. The assays were performed with 1.0 nM GrB-anti-CD19 and various concentrations of anti-CD5-VCE. For comparison, we also measured cytotoxicity of anti-CD5-VCE bearing the endogenous furin cleavage sequence (anti-CD5-VCE_(wt)) and a mutant anti-CD5-VCE in which one of the predicted active site residues glutamic acid 613 was replaced with alanine (anti-CD5-VCE_(E613A)). Nonlinear regression analysis was performed as described above.

FIG. 12A is a schematic depiction of N-GFD-VCE. For comparison, the structure of anti-CD5-VCE is also shown. N-GFD-VCE was expressed in a soluble form from E. coli, and purified by Ni-NTA affinity purification.

FIG. 12B is a graph showing cytotoxicity assay results using CD19⁺ Jurkat cells. Both N-GFD-VCE_(wt) and the combination of N-GFD-VCE_(GrB) and GrB-anti-CD19 are toxic to the target cells.

FIG. 13A, FIG. 13B, and FIG. 13C are graphs showing selective cytotoxicity of combinatorial targeting agents to CD5⁺ B cells in PBMNC from a B-CLL patient. FIG. 13A shows FACS analysis of purified PBMNC from a B-CLL patient with anti-CD5 and anti-CD19 antibodies. FIG. 13B shows 1.0 nM GrB-anti-CD19 alone was not toxic to either PBMNC or CD5⁺Raji. FIG. 13C shows that anti-CD5-VCE selectively kill CD5⁺Raji cells and a fraction of PBMNC only in the presence of GrB-anti-CD19.

FIG. 14 is a graph showing cytotoxicity assay results of a DT_(GrM)-anti-CD19 and GrM-anti-CD5 combination toward a CD19⁺Jurkat cell line. CD19⁺ Jurkat cells were treated with 2 nM of GrM-anti-CD5 and various concentrations of DT_(GrM)-anti-CD19. The presence of GrM-anti-CD5 increased the toxicity of DT_(GrM)-anti-CD19.

FIG. 15 is a graph showing selective killing of CD5⁺Raji cells using DT-anti-CD22 and GrB-anti-CD5 (anti-CD5=CT5 ScFv or MH6 ScFv) fusion proteins. Protein synthesis inhibition was analyzed by quantitation of ³[H]-leucine uptake in comparison to buffer treated controls.

FIG. 16 is a schematic depiction of anti-CD5-Aerolysin_(GrB), which is prepared from anti-CD5 ScFv (LPETGGVE SEQ ID NO:21) and GK-Aerolysin_(GrB) (GKGGSNSAAS SEQ ID NO: 22) through a ligation reaction catalyzed by S. aureus Sortase A.

FIG. 17A and FIG. 17B are photographs showing 4-20% gradient SDS-PAGE gels of aerolysin-ScFv conjugation catalyzed by Sortase A. Refolded anti-CD5 ScFv and soluble GK-Aerolysin_(GrB) were mixed (lane 1), treated with immobilized Sortase A (lane 2) or soluble Sortase A (lane 3 of FIG. 17A) and incubated at room temperature overnight. The conjugated mixture was then incubated with mouse GrB for 3 hours at room temperature (lane 3 of FIG. 17B).

FIG. 17C is a graph showing the purification profile of Sortase A conjugated anti-CD5-Aerolysin_(GrB) over a Q-anion exchange column. The purified fusion protein was concentrated and analyzed against the input material using 4-20% gradient SDS-PAGE.

FIG. 18A and FIG. 18B are graphs showing cytotoxicity assay results using aerolysin based immunotoxins. FIG. 18A illustrates the effect of GrB-anti-CD19 (2 nM) on the cytotoxicity of anti-CD5-Aerolysin_(GrB) towards CD5⁺Raji and CD19⁺Jurkat cells. FIG. 18B illustrates the effect of anti-CD5 ScFv domain for cytotoxicity, as well as the requirement of CD5 surface antigen for cytotoxicity of the combinatorial targeting reagents.

FIG. 19 is a graph showing cytotoxicity assay results using CD5⁺JVM3 and JeKo-1 cells. CD5⁺JVM3 or JeKo-1 cells were incubated with anti-CD5-aerolysin_(GrB) with or without 2 nM of GrB-anti-CD19. Anti-CD5-aerolysin_(GrB) shows toxicity to both CD5⁺JVM3 or JeKo-1 cell lines in the presence of GrB-anti-CD19. GK-Aerolysin_(GrB) is not toxic to CD5⁺JVM3 cells.

FIG. 20A is a schematic depiction of an enzymatically active GrB-(YSA)₂ fusion protein, an enterokinase activatable GrB-(YSA)₂ fusion protein DDDDK-GrB-YSA (SEQ ID NO:25), and a furin activatable RSRR-GrB-(YSA)₂ (SEQ ID NO:26) fusion protein. The amino acid sequences of the pro-domains are shown.

FIG. 20B is a graph showing that purified DDDDK-GrB-(YSA)₂ (SEQ ID NO:25) fusion protein may be activated using enterokinase. The granzyme B activity before (open circles) and after (open rectangles) enterokinase treatment are shown. The GrB activity was monitored using fluorogenic substrate Ac-IEPD-AMC.

FIG. 20C is a graph showing in vivo furin activation of the furin activatable RSRR-GrB-(YSA)₂ fusion protein. Both pro-GrB-(YSA)₂ fusion proteins were expressed in 293T cells, which naturally express furin. The fusion proteins were collected and their GrB activity measured as described above. Whereas the furin activatable RSRR-GrB-(YSA)₂ (SEQ ID NO:26) was active (open rectangles), no GrB activity was observed for the enterokinase activatable DDDDK-GrB-(YSA)2 (SEQ ID NO:25) (open circles).

FIG. 21A is a schematic depiction of various thioredoxin-DT fusion proteins containing the wild type or mutated furin cleavage site.

FIG. 21B is a photograph of an SDS PAGE gel showing the site specific cleavage of these fusion proteins by incubating with furin at 37° C. for 20 min.

FIG. 22A is a schematic showing the desired phosphorylation reactions (SEQ ID NOs:4, 29-31, from top to bottom).

FIG. 22B is an image showing the radiolabeled fusion proteins after phosphorylation using PKA and γ-³²P-ATP.

FIG. 22C shows the reaction mixtures after overnight treatment with furin at 37° C. It is evident that the phosphorylated proteins pDT^(A), PDT^(AT), and pDT^(S) are resistant to furin cleavage.

FIG. 23A is a schematic depiction of the Trx-DT^(A)-anti-CD19 fusion proteins with mutated and/or modified furin cleavage sites shown.

FIG. 23B is a graph showing that the unphosphorylated Trx-DT^(A)-anti-CD19 fusion was toxic to all the cells tested, with IC50˜0.01-0.1 nM, whereas the phosphorylated Trx-DT^(A)-anti-CD19 fusion was not toxic to these cells under similar conditions.

FIG. 24 is a schematic depiction of fusion and hybrid proteins generated to target claudin3/4 or EphA2 surface antigens overexpressed on breast cancer cells. The cell-targeting moiety of DT_(GrB)-CCPE fusion protein is C-CPE, the C-terminal domain of the Clostridium peringens enterotoxin, which binds with high affinity and specificity to the mammalian claudin3/4 adhesion molecules. The cell-targeting moiety of GrB-(YSA)₂ fusion protein is a repeat fusion of YSA peptide, which is a 12 residue peptide YSAYPDSVPMMS (SEQ ID NO:34) that can specifically recognize EphA2 receptors. Hybrid protein GrB-(YSA)₃ contains three YSA peptides linked to GrB through a branched chemical linker, to which one GrB molecule and three YSA peptides are linked through their C-terminus carboxyl group.

FIG. 25A is a schematic showing the design of fusion proteins DT-anti-CD22-anti-CD19 and GrB-anti-CD19-anti-CD19.

FIG. 25B and FIG. 25C are photographs of SDS PAGE gels showing fusion proteins DT-anti-CD22 anti-CD19 and GrB-anti-CD19-anti-CD19, each containing two fused ScFv binding motifs.

FIG. 26A is a schematic depiction of fusion protein NGFD-VCE_(TEV), which comprises a VCE based protoxin containing a TEV cleavage site in place of the native furin cleavage site and a cell-targeting moiety N-GFD for u-PAR binding.

FIG. 26B is a schematic depiction of the preparation of anti-CD5-TEV hybrid protein using S. aureus Sortase A catalyzed ligation of a LEPTG tagged anti-CD5 ScFv moiety and a GKGG tagged TEV protease.

FIG. 27A is an SDS-PAGE analysis of NGFD-VCE_(TEV) fusion protein and its cleavage in a reaction mixture Containing TEV protease. As expected, protoxin NGFD-VCE_(TEV) is specifically cleaved by TEV protease.

FIG. 27B is a graph showing cytotoxicity assay results using CD19⁺Jurkat cells (CD5⁺/uPAR⁺) treated with various concentrations of NGFD-VCE_(TEV) fusion (VCE), anti-CD5-TEV hybrid (TEV), or their mixture. The data illustrates that the combination of 15 nM of NGFD-VCE_(TEV) and 1.5 nM of anti-CD5-TEV is significantly more toxic to the CD19⁺Jurkat cells than either NGFD-VCE_(TEV) or anti-CD5-TEV alone at the same concentrations.

FIG. 28 is an SDS gel showing susceptibility of engineered VCE molecules to granzyme B. VCE_(IEPD): the native furin cleavage site RKPR is replaced by IEPD; VCE_(IAPD): the native furin cleavage site is replaced by IAPD; W: wild type GrB; T: N218T mutant of GrB.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods and compositions for treating various diseases through selective killing of targeted cells using a combinatorial targeting approach. In one aspect, the invention features protoxin fusion proteins containing a cell targeting moiety and, a modifiable activation moiety which is activated by an activation moiety not naturally operably found in, on, or in the vicinity of a target cell. These methods also include the combinatorial use of two or more therapeutic agents, at minimum comprising a protoxin and a protoxin activator, to target and destroy a specific cell population. Each agent contains at least one cell targeting moiety that binds to an independent cell surface target of the targeted cells. The protoxin contains a modifiable activation moiety that may be acted upon by the protoxin activator. The protoxin activator comprises an enzymatic activity that upon acting on the modifiable activation moiety converts, or allows to be converted, the protoxin to an active toxin or a natively activatable toxin. The targeted cells are then inhibited or destroyed by the activated toxin.

The present invention also provides for the use of multiple independent targeting events to further restrict or make selective the recognition of cells that are desired to be inhibited or destroyed, through the use of modified protoxins and protoxin activators. The protoxin activators of the invention may contain an activation domain. Prior to activation of the activation domain by a proactivator, these protoxin activators are inactive (i.e., they cannot activate the protoxin). Examples of such protoxin proactivators include proteases specific for the protoxin modifiable activation moiety that are presented in zymogen form, such that the cleavage of the zymogen to activate the proactivator requires a second protease. Examples of moieties provided by this invention include targeted granzyme B bearing an enterokinase-susceptible peptide blocking the active site, and targeted granzyme B bearing a furin-susceptible peptide blocking the active site. A suitable example of a protoxin proactivator, would be an enterokinase fusion protein that can be independently targeted to the target cell and act upon the granzyme B bearing an enterokinase-susceptible peptide blocking the active site.

The present invention also provides for the activation of protoxins or proactivators by modifiable activation moieties that allow said protoxins or proactivators to be activated or converted to a form that may be natively activated. Modifiable activation moieties may be polypeptide cleavage sequences, altered polypeptide cleavage sequences, or cleavable linkers, that restrict or make selective the activation of the protoxin or proactivator. Each modifiable activation moiety must have a corresponding activator capable of modifying such modifiable activation moiety in a way that causes the protoxins or proactivators bearing such modifiable activation moiety to be activated or converted to a form that may be natively activated.

I. Disease Indications and Targeted Cell Surface Markers

The protoxin/toxin activator combinations of the invention target and kill specific cell subsets while sparing closely related cells. The utility of the invention lies in the selective elimination of subsets of cells to achieve a desired therapeutic effect. In particular the combinations of the present invention can target cancer cells while sparing closely related normal cells, thereby providing a more specific and effective treatment for cancer. The cell-targeting moieties can target cell surface targets on the targeted cancer cells, or on targeted noncancer cells that are preferably eliminated to achieve a therapeutic benefit.

A. Cell Surface Targets

One or both of the cell-targeting moieties can target a cell surface target typical of a specific type of cells, for example, by recognizing lineage-specific markers found on subsets of cells and representing their natural origin, such as markers of the various organs of the body or specific cell types within such organs, or cells of the hematopoietic, nervous, or vascular systems. Alternatively one or both of the cell-targeting moieties can target cell surface markers aberrantly expressed on a diseased tissue, such as a cancer cell or a cell eliciting or effecting an autoimmune activity (e.g., B cells, T cells, dendritic cells, NK cells, neutrophils, leukocytes, macrophages, platelets, macrophages, myeloid cells, and granulocytes). One or both agents can target a cell surface marker that is aberrantly overexpressed by a cancer cell. This multi-agent targeting strategy is used to target neoplastic or undesired cells selectively without severe damage to normal or desired cells, thereby providing treatments for cancers including leukemias and lymphomas, such as chronic B cell leukemia, mantle cell lymphoma, Acute myelogenous leukemia, chronic myelogenous leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, multiple myeloma, acute lymphoblastic leukemia, adult T-cell leukemia, Hodgkin's lymphoma, and non-Hodgkin's lymphoma; as well as solid tumors, including melanoma, colon cancer, breast cancer, prostate cancer, ovarian cancer, lung cancer, pancreatic cancer, kidney cancer, stomach cancer, liver cancer, bladder cancer, thyroid cancer, brain cancer, bone cancer, testicular cancer, uterus cancer, soft tissue tumors, nervous system tumors, and head and neck cancer.

The combination of protoxin and protoxin activator proteins can also be used to target non-cancerous cells, including autoreactive B or T cells, providing treatment for chronic inflammatory diseases including multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, Sjogren's syndrome, scleroderma, primary biliary cirrhosis, Graves' disease, Hashimoto's thyroiditis, type 1 diabetes, pernicious anemia, myasthenia gravis, Reiter's syndrome, immune thrombocytopenia, celiac disease, inflammatory bowel disease, and asthma and atopic disorders.

In addition the combinatorial therapeutic composition can be used to ablate cells in the nervous system that are responsible for pathological or undesired activity, for example nociceptive neurons in the peripheral nervous system, or to treat sensory phantom sensation, or to control neuropathic pain, such as the pain caused by diabetic neuropathy or viral reactivation.

The combination can also target cells infected by viral, microbial, or parasitic pathogens that are difficult to eradicate, providing treatment for acquired syndromes such as HIV, HBV, HCV or papilloma virus infections, tuberculosis, malaria, dengue, Chagas' disease, trypanosomiasis, leishmaniasis, or Lyme disease.

Furthermore, the combination can target specific cell types including, without limitation, parenchymal cells of the major organs of the body, as well as adipocytes, endothelial cells, cells of the nervous system, pneumocytes, B cells or T cells of specific lineage, dendritic cells, NK cells, neutrophils, leukocytes, macrophages, platelets, macrophages, myeloid cells, granulocytes, adipocyte, and any other specific tissue cells.

The combination can further target cells that produce disease through benign proliferation, such as prostate cells in benign prostatic hypertrophy, or in various syndromes leading to hyperproliferation of normal tissues or the expansion of undesired cellular compartments as for example of adipocytes in obesity.

It will be well recognized by those skilled in the art that there are many cell surface targets that may be used for targeting the protoxins or protoxin activators of the invention to tumor tissues. For example, breast cancer cells may be targeted using overexpressed surface antigens such as claudin-3 (Soini, Hum. Pathol. 35:1531 (2004)), claudin-4 (Soini, Hum. Pathol. 35:1531 (2004)), MUC1 (Taylor-Papadimitriou et al., J. Mammary Gland Biol. Neoplasia 7:209 (2002)), EpCAM (Went et al., Hum. Pathol. 35:122 (2004)), CD24 (Kristiansen et al., J. Mol. Histol. 35:255 (2004)), and EphA2 (Ireton and Chen, Curr. Cancer Drug Targets 5:149 (2005); Zelinski et al., Cancer Res. 61:2301 (2001)), as well as HER2 (Stem, Exp. Cell Res. 284:89 (2003)), EGFR (Stern, Cell Res. 284:89 (2003)), CEA, and uPAR (Han et al., Oncol. Rep. 14:105 (2005)). Colorectal cancer may be targeted using upregulated surface antigens such as A33 (Sakamoto et al., Cancer Chemother. Pharmacol. 46:S27 (2000)), EpCAM (Went et al., Hum. Pathol. 35:122 (2004)), EphA2 (Ireton and Chen, Curr. Cancer Drug Targets 5:149 (2005); Kataoka et al., Cancer Sci. 95:136 (2004)), CEA (Hammarstrom, Semin. Cancer Biol. 9:67 (1999)), CSAp, EGFR (Wong, Clin. Ther. 27:684 (2005)), and EphB2 (Jubb et al., Clin. Cancer Res. 11:5181 (2005)). Non-small cell lung cancer may be targeted using EphA2 (Kinch et al., Clin. Cancer Res. 9:613 (2003)), CD24 (Kristiansen et al., Br. J. Cancer 88:231 (2003)), EpCAM (Went et al., Hum. Pathol. 35:122 (2004)), HER2 (Hirsch et al., Br. J. Cancer 86:1449 (2002)), and EGFR (Dacic et al., Am. J. Clin. Pathol. 125:860 (2006)). Mesothelin has been targeted by a PEA based immunotoxin for the treatment of NSCLC (Ho et al., Clin. Cancer Res. 13(5):1571 (2007)). Ovarian cancer may be targeted using upregulated claudin-3 (Morin, Cancer Res. 65:9603 (2005)), claudin-4 (ibid.), EpCAM (Went et al., Hum. Pathol. 35:122 (2004)), CD24 (Kristiansen et al., J. Mol. Histol. 35:255 (2004)), MUC1 (Feng et al., Jpn. J. Clin. Oncol. 32:525 (2002)), EphA2 (Ireton and Chen, Curr. Cancer Drug Targets 5:149 (2005)), B7-H4 (Simon et al., Cancer Res. 66:1570 (2006)), and mesothelin (Hassan et al., Appl. Immunohistochem Mol. Morphol. 13:243 (2005)), as well as CXCR4 (Jiang et al., Gynecol. Oncol. 20:20 (2006)) and MUC16/CA125. Pancreatic cancer may be targeted using overexpressed mesothelin (Rodriguez et al., World J. Surg. 29:297 (2005)), PSCA (Rodriguez et al., World J. Surg. 29:297 (2005)), CD24 (Kristiansen et al., J. Mol. Histol. 35:255 (2004)), HER2 (Garcea et al., Eur. J. Cancer 41:2213 (2005)), and EGFR (Garcea et al., Eur. J. Cancer 41:2213 (2005)). Prostate cancer may be targeted using PSMA (Kinoshita et al., World J. Surg. 30:628 (2006)), PSCA (Hari et al., J. Urol. 171:1117 (2004)), STEAP (Hubert et al., Proc. Natl. Acad. Sci. USA 96:14523 (1999)), and EphA2 (Ireton and Chen, Curr. Cancer Drug Targets 5:149 (2005)). EpCAM is also upregulated in prostate cancer and has been targeted for its antibody-based treatment (Oberneder et al., Eu. J. Cancer 42:2530 (2006)). The expression of activated leukocyte cell adhesion molecule (ALCAM, as known as CD166) is a prognostic and diagnostic marker for prostate cancer (Kristiansen et al., J. Pathol. 205:359 (2005)), colorectal cancer (Weichert et al., J. Clin. Pathol. 57:1160 (2004)), and melanoma (van Kempen et al. Am. J. Pathol. 156(3):769 (2000)). All cancers that have been treated with chemotherapy and developed multidrug resistance (MDR) can be targeted using the transmembrane transporter proteins involved, including P-glycoprotein (P-gp), the multidrug resistance associated protein (MRP1), the lung resistance protein (LRP), and the breast cancer resistance protein (BCRP) (Tan et al., Curr. Opin. Oncol. 12:450 (2000)). Any of the above markers may be targeted by the fusion proteins of the invention.

Significant advances have been made during the past decade in the identification of unique cell surface marker profiles of cancer stem cells from various cancers, distinguishing them from the bulk of corresponding tumor cells. For example, in acute myeloid leukemia (AML) it has been observed that the CD133+/CD38−. AML cells, which constitute a small fraction of CD34+/CD38− AML cells, are responsible for initiating human AML in animal models (Yin et al., Blood 12:5002 (1997)). In addition, CD133 has been recently determined as a cancer stem cell surface marker for several solid tumors as well, including brain tumor (Singh et al., Nature 432:395 (2004) and Bao et al., Nature 444:756 (2006)), colon cancer (O'Brien et al., Nature 445:106 (2007) and Ricci-Vitiani et al, Nature 445:111 (2007)), prostate cancer (Rizzo et al., Cell Prolif. 38:363 (2005)), and heptocellular carcinoma (Suetsugu et al., Biochem. Biophys. Res. Commun. 351:820 (2006) and Yin et al., Int. J. Cancer 120:1444 (2007)). In the case of colon cancer, the CD133+ tumorgenic cells were found to bind antibody Ber-EP4 (Ricci-Vitiani et al, Nature 445:111 (2007)), which recognizes the epithelial cell adhesion molecules (EpCAM), also known as ESA and CD326. More recently, it was reported that CD44+ may more accurately define the CSC population of colorectal cancer than CD133+ does, and the CSCs for colorectal cancer have been identified as EpCAM^(high)/CD44+/CD166+ (Dalerba et al., Proc. Natl. Acad. Sci. USA 104(24):10158 (2007)). Based on this information, EpCAM/CD133, EpCAM/CD44, EpCAM/CD166, and CD44/CD166 are possible combinations for combinatorial targeting of colon cancer CSCs. In addition to CD133, prostate cancer stem cells have been separately identified to be CD44+ (Gu et al. Cancer Res. 67:4807 (2007)), thus they may be targetable by using the CD44/CD133 pair of surface markers. Furthermore, CXCR4 was detected in the CD44+/CD133+ putative prostate CSCs, suggesting that the combination of CXCR4 with either CD44 or CD133 may provide useful pairs of targets for combinatorial targeting strategy. In other CSCs where the only currently known surface antigen is CD133, additional surface antigens may be identified through comprehensive antibody screening and then used to complement CD133 in a combinatorial targeting scheme. Likewise, tumorigenic cells for breast cancer have been identified as CD44+/CD24− subpopulation of breast cancer cells. Further analysis revealed that the CD44+/CD24−/EpCAM+ fraction has even higher tumorigenicity (Al-Hajj et al., Proc. Natl. Acad. Sci. USA 100:3983 (2003)). A combinatorial targeting approach using CD44+ and EpCAM+ as targeted surface markers could specifically kill these CSCs while leaving normal CD44+ leukocytes/erythrocytes and normal EpCAM+ epithelial cells unharmed. Another recent study has shown that pancreatic CSCs are CD44+/CD24+/EpCAM+ (Li et al., Cancer Res. 67:1030 (2007)). Consequently, the pancreatic CSCs may be targeted using a combination of CD44/CD24, CD44/EpCAM, or CD24/EpCAM.

B cell chronic lymphocytic leukemia (B-CLL) is characterized by slowly accumulating CD5⁺ B cells (Guipaud et al., Lancet Oncol. 4:505 (2003)). CD5 is a cell surface protein found on normal T cells and a small fraction of B cells, known as B1 cells. Immunotoxins that target CD5 have shown high efficacy in killing T cells (Better et al., J. Biol. Chem. 270:14951 (1995)). The combinatorial targeting strategy described in this invention makes it possible to use CD5 in combination with a B cell marker such as CD19, CD20, CD21, or CD22, thereby distinguishing B-CLL cells or other B cells in the B1 subset from T cells. The B1 subset is thought to give rise to low affinity polyreactive antibodies that are frequently found in the setting of autoimmune disorders, hence ablation of this population without significantly impairing the remainder of B cells could favorably impact the course of autoimmune disease without comprising the immune response of an individual to the same extent that ablation of all B cells would induce.

Examples of combinations of surface antigens that can be useful targets for the protoxin activator (e.g., protease) fusion and toxin fusion proteins of the invention are set forth in Table 1.

TABLE 1 Antigen Target Normal Cancer Targeted Antibody Antibody ScFv Pair Antigen Availability Distribution Marker Cells Sequences Immunotoxins Immunotoxins Targeted Cancer: Breast Cancer [Claudin- Claudin-3 Abnova Tight junctions at Expression in 92-100% Carcinoma C-terminal None C-CPE-PEA 3 & 4]/ Claudin-4 Corporation: the apical junctional of breast cells domain of C. perfringens fusion: [EpCAM] H00001365-P01 complex in carcinomas, enterotoxin (C- J Pharmacol Exp [Caludin- (claudin-3) epithelial and claudin-3 and -4 CPE) can bind Ther. 2006, 3 & 4]/ H00001364-Q01 endothelial overexpressed in claudin-3 and - 316(1): 255 [EphA2] (Claudin-4) cellular sheets; 62% or 26% of 4 specifically [Claudin- gut, lungs, and breast carcinomas, 3 & 4]/ kidneys respectively [MUC1] MUC1 Abnova Expressed at the Expression in Breast Cancer Immunol Calicheamicin Ribonuclease Etc. (Mucin 1) Corporation: luminal surface ~90% breast carcinoma Immunother. 1999, conjugate: Bioconjug fusion: Br J Cancer. H00004582- of most simple carcinomas; cells 48(1): 29 Chem. 2005, 2004, 90(9): 1863 Q01 epithelial cells correlates with Mol Immunol. 16(2): 346 & 354 (partial lower grade 2005, 42(1): 55 sequence) tumors U.S. Pat. No. 6,506,881 (V_(H) & V_(L)) EpCAM R&D Expressed on the Upregulated in Epithelial Cancer Immunol IL2 fusion: β-glucuronidase (Epithelial Systems: baso-lateral cell ~35% breast cells and Immunother. J Immunother. fusion: Br J cell adhesion 960-EP-050 surface in most carcinomas, and breast 2001, 50(1): 51. 2004, 27(3): 211 Cancer. 2002, molecule) human simple by Taxol or cancer cells Cancer Res. 1999 86(5): 811 epithelia Navelbine; IHC 59(22): 5758 positive in 74% (V_(H) & V_(L)) samples; >100- fold increase in mRNA; correlates w/ poor prognosis EphA2 R&D Weak or negative Overexpressed in Breast Methods. 2005, None None; (Ephrin Systems: IHC in normal ~92% of breast cancer cells 36(1): 43 Ephrin memetic receptor A2) 3035-A2-100 breast tissues tumor cells (by (B233: V_(H) & V_(L)) peptides can be IHC, diffused into Mol. Immunol phage selected to cytoplasm); certain 2007, 44: 3049 bind EphA2 epitopes more (EA2 & 47: specifically exposed than in V_(H) & V_(L)) normal cells HER2 R&D Liver, kidneys, Upregulated in HER2+ Biochemistry Herceptin- PEA fusion: Systems: spleen, etc. ~20-30% breast cells 1994, 33: 5451 geldanamycin J Biol Chem. 1994, 1129-ER-050 Br J Pharmacol. cancer; correlates (dcFv V_(H) & V_(L)) conjugate: 269(28): 18327. 2004, 143(1): 99 w/ poor prognosis; J Mol Biol. Cancer Res. 2004 Breast Cancer Res only partially 1996, 255(1): 28 64(4): 1460 Treat. 2003, overlaps with (scFv V_(H) & V_(L)) 82(3): 155. EpCAM GrB fusion: overexpression Cell Death Differ. 2006 13(4): 576. EGFR R&D Kidneys, liver, Only positive in EGFR+ Int J Cancer. Taxol conjugate: PEA fusion: (Epidermal Systems: intestine, bone, ~10% breast cells 1995, 60: 137 Bioconjug Chem. Int J Cancer. 2000, growth 1095-ER-002 etc. cancer tissue (V_(H) & V_(L)) 2003, 14(2): 302 86(2): 269. factor J Nucl Med. Jpn J Cancer Res. Methotrexate GrB-TGFα fusion: receptor) 2006, 47(6): 1023 2000 91(10): 1035 conjugate: Mol Cell Death Differ. (vIII V_(H) & V_(L)) Cancer Ther. 2006, 2006 13(4): 576. 5(1): 52 CEA ProSpec-Tany Limited tissue Overexpressed in Breast Immunotech. Doxorubicin PEA fusion: Clin (Carcino- TechnoGene distribution: gastro-intestinal, carcinoma 1996, 2: 181 conjugate: Cancer Cancer Res. 1998, embryonic Ltd: colon, neck, breast, & lung cells (V_(H) & V_(L)) Immunol 4(11): 2825 antigen) PRO-287 stomach, tohue cancers; upregulated U.S. Pat. No. 2,316,2709A1 Immunother. 1994, GenScript esophagus, by drugs; also a U.S. Pat. No. 2,524,4333A1 38(2): 92 Corporation: cervix, prostate serum marker; Z00239 detected in only 19% of breast cancers uPAR R&D Low expression Overexpressed Breast U.S. Pat. No. 5,891,664 None None Systems: in normal breast by leukemias carcinoma 807-UK-100; tissue and breast cancer cells 807-UK-100/CF CD24 Abnova B cells, High IHC staining Normal B None Ricin A conjugate: None (aka HSA: Corporation: granulocytes in 85% breast cells and Int J Cancer. 1996, Heat stable H00000934-P01 cancer carcinoma 66(4): 526 antagen) cells p-Glyco- Abnova Low expression Upregulated after Drug- MRK-16: Biol PEA conjugate: PEA fusion: protein Corporation: chemotherapy resistant Chem. 1999, J Urol. 1993, Int J Cancer. 2001. (MDR1 gene H00005243- cancer cells 274(39): 27371 149(1): 174 94(6): 864 product) Q01 C219: J Biol (partial Chem. 1997, sequence) 272(47): 29784 Targeted Cancer: Colorectal Cancer (CRC) [A33]/ A33 N/A Epithelia of Carcinomas of Colorectal J Biol Chem. Carboxypeptidase Cytosine-deaminase [EGFR- Recombinant gastrointestinal the colon and epithelial 2000, A fusion: fusion: Br J Cancer. HER2] expression in tract (colonic, rectum; a cells 275(18): 13668 Int J Oncol. 2004, 2003, 88(6): 937. [A33]/ insect cells: small intestinal, glycoprotein (V_(H) & V_(L)) 24(5): 1289 Pichia expression of [CEA] Biotechnol Prog. and duodenal found in 95% scFv: Protein Expr. [A33]/ 2004, epithelium) CRC cancers Purif. 2004, 37: 18 [CD15] 20(4): 1273 [EpCAM]/ EpCAM R&D Expressed on the Upregulated in Colorectal Cancer Immunol IL2 fusion: β-glucuronidase [EGFR- (Epithelial Systems: baso-lateral cell colon epithelia; epithelial Immunother. J Immunother. fusion: Br J HER2] cell 960-EP-050 surface in most upregulated by cells 2001, 50(1): 51 2004, 27(3): 211 Cancer. 2002, Etc. adhesion human simple Taxol and Cancer Res. 86(5): 811 molecule) epithelia Navelbine; IHC 1999 positive in 100% 59(22): 5758 tissue samples (V_(H) & V_(L)) EphA2 R&D Some expression Upregulated in 50-70% Colon Methods. 2005, None None; (Ephrin Systems: in normal colon of primary cancer cells 36(1): 43 Ephrin memetic receptor 3035-A2-100 tissue colorectal tumor (V_(H) & V_(L)) peptides can be A2) cells (IHC); phage selected to downregulated in bind EphA2 metastasis specifically CEA ProSpec-Tany Limited tissue Overexpressed in Colorectal Immunotech. Doxorubicin PEA fusion: Clin (Carcino- TechnoGene distribution: many cancers, e.g., epithelial 1996, 2: 181 conjugate: Cancer Cancer Res. 1998, embryonic Ltd: colon, neck, gastrointestinal, cells (V_(H) & V_(L)) Immunol 4(11): 2825 antigen) PRO-287 stomach, tohue, breast, and lung Colorectal U.S. Pat. No. 2,316,2709A1 Immunother. 1994, GenScript esophagus, cancers. Can be carcinoma U.S. Pat. No. 2,524,4333A1 38(2): 92 Corporation: cervix, prostate further upregulated cells Z00239 by drugs. Elevated levels in serum. CD15 N/A Neutrophils, Expressed in CRC, CEA+ and Proc Natl Acad None None (Sialyl eosinophiles, AML, and other EpCAM+ Sci USA. 1999, lewis X) monocytes cancers; correlated CRC cells 96(12): 6953 with EpCAM+ and (scFv V_(H) & V_(L)) CEA+ CRC cells: U.S. Pat. No. 5,723,583A2 Proteomics. 2006, 6(6): 1791 CSAp N/A Restricted to the Present in 60% Colorectal Cancer. 1997, ¹³¹I conjugate: None (Colon intestines colorectal carcinoma 80(12 Cancer. 1994, 73(3 specific carcinomas cells Suppl): 2667 Suppl): 864- antigen-p) CD166 R&D Broad distribution, Strong cell Epithelial Reported in J. None Saporin S6 (ALCAM: Systems: in epithelia, surface cells and Cell Biol. 2005, conjugate: J. Cell Activated 656-AL neurons, lymphoid expression in other normal 118(7): 1515 & Biol. 2005, leukocyte and myeloid cells, 31% colorectal cells, and Liu B., et al. J. 118(7): 1515 cell hematopoietic and carcinoma; colorectal Mol. Med. 2007, adhesion mesenchymal stem mRNA cancer cells but sequences molecule) cells overexpression were not in 86% prostate disclosed carcinoma EGFR R&D Kidneys, liver, Upregulated in EGFR+ Int J Cancer. Taxol conjugate: PEA fusion: (Epidermal Systems: intestine, bone, cancers of colon, cancer cells 1995, 60: 137 Bioconjug Chem. Int J Cancer. 2000, growth 1095-ER-002 etc. breast, etc. EGFRvIII (V_(H) & V_(L)) 2003, 14(2): 302 86(2): 269. factor J Nucl Med. Level correlates mutant in Jpn J Cancer Methotrexate GrB-TGFα fusion: receptor) 2006, 47(6): 1023 with tumor PCa Res. 2000 conjugate: Mol Cell Death Differ. progression 91(10): 1035 Cancer Ther. 2006, 2006 13(4): 576. (vIII V_(H) & V_(L) ) 5(1): 52 HER2 R&D Liver, kidneys, Upregulated in HER2+ Biochemistry Herceptin- PEA fusion: Systems: spleen, etc. cancers of colon, cancer cells 1994, 33: 5451 geldanamycin J Biol Chem. 1994, 1129-ER-050 Br J Pharmacol. breast, etc. (dcFv V_(H) & conjugate: 269(28): 18327. 2004, 143(1): 99 V_(L)) Cancer Res. 2004 Breast Cancer Res J Mol Biol. 64(4): 1460 Treat. 2003, 1996, 82(3): 155. 255(1): 28 GrB fusion: (V_(H) & V_(L) ) Cell Death Differ. 2006 13(4): 576. EGFR- See above Advantages of bispecific targeting: not EGFR+ or US20060099205 None Bivalent PEA fusion: HER2 limited by a single marker and higher HER2+ A1: Bispecific Br J Cancer. 1996, target density, neither is achievable by cancer cells single chain FVs 74(6): 853. natural protease system, e.g., uPA/uPAR (V_(H) & V_(L)) Int J Cancer. 1996, 65(4): 538: p-Glyco- Abnova Low expression Upregulated after Drug- MRK-16: Biol PEA conjugate: PEA fusion: protein Corporation: chemotherapy resistant Chem. 1999, J Urol. 1993, Int J Cancer. 2001, (MDR1 H00005243-Q01 cancer cells 274(39): 27371 149(1): 174 94(6): 864 gene (partial C219: J Biol product) sequence) Chem. 1997, 272(47): 29784 Targeted Cancer: Non-Small Cell Lung Cancer (NSCLC) [EphA2]/ EphA2 R&D Overexpressed NSCLC Methods. 2005, None None; [CD24] (Ephrin Systems: in ~74% cells 36(1): 43 Ephrin memetic [EphA2]/ receptor A2) 3035-A2-100 (moderate-high) (V_(H) & V_(L)) peptides can be [EpCAM] and detectable in phage selected to etc. 96% of NSCLC bind EphA2 tissue (by IHC, specifically in cytoplasm and membrane) CD24 Abnova B cells, ~40-60% of Normal B None Ricin A conjugate: None (aka HSA: Corporation: granulocytes cancer tissue cells and Int J Cancer. 1996, Heat stable H00000934-P01 samples with carcinoma 66(4): 526 antagen) (full length) high IHC cells staining; higher expression level corresponds to poor prognosis EpCAM R&D Expressed on the IHC positive in Cancer Immunol IL2 fusion: β-glucuronidase (Epithelial Systems: baso-lateral cell 92% tissue Immunother. J Immunother. fusion: Br J cell 960-EP-050 surface in most samples 2001, 50(1): 51 2004, 27(3): 211 Cancer. 2002, adhesion human simple Cancer Res. 86(5): 811 molecule) epithelia 1999 59(22): 5758 (V_(H) & V_(L)) HER2 R&D Liver, kidneys, Overexpression HER2+ Biochemistry Herceptin- PEA fusion: Systems: spleen, etc. in 16% and cancer cells 1994, 33: 5451 geldanamycin J Biol Chem. 1994, 1129-ER-050 Br J Pharmacol. detection in 43% (dcFv V_(H) & V_(L)) conjugate: 269(28): 18327. 2004, 143(1): 99 NSCLC tumor J Mol Biol. Cancer Res. 2004 Breast Cancer Res samples 1996, 255(1): 28 64(4): 1460 Treat. 2003, 82(3): 155. (V_(H) & V_(L)) GrB fusion: Cell Death Differ. 2006 13(4): 576. EGFR R&D Kidneys, liver, Detection in 11-26% EGFR+ Int J Cancer. Taxol conjugate: PEA fusion: Systems: intestine, bone, NSCLC cancer cells 1995, 60: 137 Bioconjug Chem. Int J Cancer. 2000, 1095-ER-002 etc. tissue samples (V_(H) & V_(L)) 2003, 14(2): 302 86(2): 269. J Nucl Med. Jpn J Methotrexate GrB-TGFα fusion: 2006, 47(6): 1023 Cancer conjugate: Mol Cell Death Differ. Res. 2000 Cancer Ther. 2006, 2006 13(4): 576. 91(10): 1035 5(1): 52 (vIII V_(H) & V_(L)) EGFR- See above Advantages of bispecific targeting: EGFR+ or US20060099205 None Bivalent PEA fusion: HER2 not limited by a single marker and HER2+ A1: Bispecific Br J Cancer. 1996, higher target density, neither is cancer cells single chain FVs 74(6): 853. achievable by natural protease system, (V_(H) & V_(L)) Int J Cancer. 1996, e.g., uPA/uPAR 65(4): 538. MSLN Abnova Methothelial cells; Upregulated for Lung cancer J Mol Biol. PEA conjugate: PEA fusion: (Mesothelin) Corporation: Stomach, >16-fold in cells, 1998, J Immunother. 2000, J Mol Biol. 1998, H00010232-Q01 peritoneum, and pancreatic methothelial 281(5): 917 23(4): 473 281(5): 917 (partial ovary cancer tissues cells (V_(H) & V_(L)) sequence) and cell lines; Mol. Immunol. detected in 1997, 34(1): 9 100% patients (V_(H) & V_(L)) p-Glyco- Abnova Low expression Upregulated Drug- MRK-16: PEA conjugate: PEA fusion: protein Corporation: after resistant Biol Chem. 1999, J Urol. 1993, Int J Cancer. 2001, (MDR1 H00005243-Q01 chemotherapy cancer cells 274(39): 27371 149(1): 174 94(6): 864 gene (partial C219: product) sequence) J Biol Chem. 1997, 272(47): 29784 Targeted Cancer: Ovarian Cancer [Claudin- Claudin-3 Abnova Tight junctions at Claudin-3 Ovarian C-terminal None C-CPE-PEA 3 & 4]/ Claudin-4 Corporation: the apical junctional upregulated in cancer cells domain of C. perfringens fusion: [EpCAM] H00001365-P01 complex in ovarian enterotoxin (C- J Pharmacol Exp [Claudin- (claudin-3, full epithelial and cancers for ~2-10 CPE) can bind Ther. 2006, 3 & 4]/ length) endothelial cellular fold claudin-3 and -4 316(1): 255 [CD24] H00001364-Q01 sheets; gut, lungs, specifically [MUC1]/ (Claudin-4, full and kidneys; low [EpCAM] length) claudin-3 in [EpCAM]/ normal ovarian [CA125- tissue B7-H4] EpCAM R&D Expressed on the Highly Epithelial Cancer Immunol IL2 fusion: β-glucuronidase Etc. (Epithelial Systems: baso-lateral cell upregulated in cells and Immunother. J Immunother. fusion: Br J cell 960-EP-050 surface in most ovarian cancer, ovarian 2001, 50(1): 51. 2004, 27(3): 211 Cancer. 2002, adhesion human simple breast cancer, cancer cells Cancer Res. 86(5): 811 molecule) epithelia, very etc; in 100% 1999 low exoression ovarian cancer 59(22): 5758 in normal ovaries tissue samples (V_(H) & V_(L)) CD24 Abnova B cells, Highly Normal B N/A Ricin A conjugate: None (aka HSA: Corporation: granulocytes upregulated cells and Int J Cancer. 1996, Heat stable H00000934- mRNA in carcinoma 66(4): 526 antagen) P01 ovarian cancer; cells (full length) IHC positive in 75-91% ovarian tumors MUC1 Abnova Expressed at the IHC positive in Ovarian Cancer Immunol Calicheamicin Ribonuclease (mucin 1) Corporation: apical surface of 100% serous cancer cells Immunother. conjugate: fusion: Br J H00004582- most simple and 75% 1999, 48(1): 29 Bioconjug Chem. Cancer. 2004, Q01 epithelia mucinous Mol Immunol. 2005, 16(2): 346 & 90(9): 1863 (partial ovarian 2005, 42(1): 55 354 sequence) carcinomas; U.S. Pat. No. 6,506,881 correlates with (V_(H) & V_(L)) higer grade ovarian cancer EphA2 R&D Little to none Upregulated in Ovarian Methods. 2005, None None; (Ephrin Systems: IHC staining in ~76% of cancer cells 36(1): 43 Ephrin memetic receptor A2) 3035-A2-100 normal ovarian ovarianl tumor (V_(H) & V_(L)) peptides can be tissue cells judging by Mol. Immunol phage selected to IHC 2007, 44: 3049 bind EphA2 (EA2 & 47: specifically V_(H) & V_(L)) B7-H4 Abnova Tightly controled Highly B7-H4+ T N/A None None Corporation: in normal upregulated in cells, dentric Mouse B7-H4 tissues: no 85-100% cells, B cells, 2154-B7-050 detection ovarian cancer macrophage, 91% homologous tissue; a serum & ovarian to human marker that cancer cells extracellular seems to sequence complement CA125 MSLN Abnova Methothelial cells; Upregulated in Ovarian J Mol Biol. PEA conjugate: PEA fusion: (Mesothelin) Corporation: Stomach, ovarian cancer cancer cells, 1998, J Immunother. 2000, J Mol Biol. 1998, H00010232-Q01 peritoneum, and methothelioma; methothelial 281(5): 917 23(4): 473 281(5): 917 (partial ovary upregulated in ~70% cells (V_(H) & V_(L)) sequence) serous Mol. Immunol. cancer 1997, 34(1): 9 (V_(H) & V_(L)) CXCR4 Abnova Expressed in Ovarian U.S. Pat. No. 7,005,503 None None Corporation: 60-70% cancer cells H00007852-Q01 ovarian (partial cancers sequence) MUC16/ Sigma- Expressed on Upregulated Hybridoma Daunorubicin IL6 fusion: CA125 Aldrich: mesothelial cells mRNA in 84% 1997, 16(1): 47 conjugate: Cancer Res. 2003, O6008 in fetal coelomic ovarian cancer (V_(H) & V_(L)) Gynecol Oncol. 63(12): 3234 (from human epithelium and tissues; but IHC 1989, 34(3): 305 fluids) its derivatives in equally positive the fetus and the for both normal adult & cancer tissues p-Glyco- Abnova Low expression Upregulated Drug- MRK-16: Biol PEA conjugate: PEA fusion: protein Corporation: after resistant Chem. 1999, J Urol. 1993, Int J Cancer. 2001, (MDR1 H00005243-Q01 chemotherapy cancer cells 274(39): 27371 149(1): 174 94(6): 864 gene (partial C219: J Biol product) sequence) Chem. 1997, 272(47): 29784 Targeted Cancer: Pancreatic Cancer [MSLN]/ MSLN Abnova Methothelial cells; Upregulated for Pancreatic J Mol Biol. PEA conjugate: PEA fusion: [PSCA] (Mesothelin) Corporation: Stomach, >16-fold-in cancer cells, 1998, J Immunother. 2000, J Mol Biol. 1998, Etc. H00010232-Q01 peritoneum, and pancreatic cancer methothelial 281(5): 917 23(4): 473 281(5): 917 (partial ovary tissues and cell cells (V_(H) & V_(L)) sequence) lines; detected in Mol. Immunol. 100% patients 1997, 34(1): 9 (V_(H) & V_(L)) PSCA Abnova Prostate:kidney = Upregulated for Pancreatic U.S. Pat. No. 06/824,780 Maytansinoid None (Prostate Corporation: 4084:152 per >16-fold in cancer cells conjugate: Cancer stem cell H00008000- 10k actin mRNA Pancreatic cell Res. 2002, 62: 2546 antigen) Q01 (partial lines sequence) Claudin4 Abnova Lung, breast, mRNA Pancreatic C-terminal None C-CPE-PEA fusion: Corporation: colon upregulated for cancer cells domain of C. perfringens J Pharmacol Exp H00001364- >32-fold in enterotoxin (C- Ther. 2006, Q01 pancreatic cell CPE) can bind 316(1): 255 (full length) lines; no IHC specifically observation CD24 Abnova B cells, IHC positive in Normal B N/A Ricin A conjugate: None Corporation: granulocytes 72% pancreatic cells and Int J Cancer. 1996, H00000934- tumors carcinoma 66(4): 526 P01 cells (full length) EGFR R&D Kidneys, liver, Upregulated in ~ EGFR+ Int J Cancer. Taxol conjugate: PEA fusion: Systems: intestine, bone, 31-68% cancer cells 1995, 60: 137 Bioconjug Chem. Int J Cancer. 2000, 1095-ER-002 etc. pancreatic cancer (V_(H) & V_(L)) 2003, 14(2): 302 86(2): 269. J Nucl Med. patients Jpn J Methotrexate GrB-TGFα fusion: 2006, 47(6): 1023 Cancer conjugate: Mol Cell Death Differ. Res. 2000 Cancer Ther. 2006, 2006 13(4): 576. 91(10): 1035 5(1): 52 (vIII V_(H) & V_(L)) HER2 R&D Liver, kidneys, Upregulated in ~ HER2+ Biochemistry Herceptin- PEA fusion: Systems: spleen, etc. 28% pancreatic cancer cells 1994, 33: 5451 geldanamycin J Biol Chem. 1994, 1129-ER-050 Br J Pharmacol. cancer patients (dcFv V_(H) & V_(L)) conjugate: 269(28): 18327. 2004, 143(1): 99 J Mol Biol. Cancer Res. 2004 Breast Cancer Res 1996, 255(1): 28 64(4): 1460 Treat. 2003, (V_(H) & V_(L)) 82(3): 155. GrB fusion: Cell Death Differ. 2006 13(4): 576. EGFR- See above Advantages of bispecific targeting: not EGFR+ or US20060099205 None Bivalent PEA fusion: HER2 limited by a single marker and higher HER2+ A1: Bispecific Br J Cancer. 1996, target density, neither is achievable by cancer cells single chain FVs 74(6): 853. natural protease system, e.g., uPA/uPAR (V_(H) & V_(L)) Int J Cancer. 1996, 65(4): 538. p-Glyco- Abnova Low expression Upregulated after Drug- MRK-16: Biol PEA conjugate: PEA fusion: protein Corporation: chemotherapy resistant Chem. 1999, J Urol. 1993, Int J Cancer. 2001, (MDR1 H00005243-Q01 cancer cells 274(39): 27371 149(1): 174 94(6): 864 gene (partial C219: J Biol product) sequence) Chem. 1997, 272(47): 29784 Targeted Cancer: Prostate Cancer (Pca) [STEAP]/ PSMA N/A Prostate:liver:kidney = Upregulated in Prostate U.S. Pat. No. 07/045,605 (1) Maytansinoid PEA fusion: [PSCA] (Prostate Baculovirus 174:14:11 per higher grade Pca; epithelial (V_(H) & V_(L)) conjugate: Cancer Cancer Immunol. [STEAP]/ specific expression 10k actin mRNA; Strong IHC cells Res. 2004, 64: 7995 Immunother. 2006 [PSMA- membrane described in Strong IHC stain stain for 8/19 (apically (2) Ricin A fusion: pub on web PSCA] antigen) Protein Expr for 15/23 prostate, prostate samples. localized) Prostate 2004, 61: 1 [PSMA/ Purif. 2000, 22/22 kidney, & (Apical PSCA] 19(1): 12 11/18 bladder localization) [PSCA/ samples EphA2] PSCA Abnova Prostate:kidney = Detected in Prostate U.S. Pat. No. 06/824,780 Maytansinoid None Etc. (Prostate Corporation: 4084:152 per 94% Pca epithelial conjugate: Cancer stem cell H00008000- 10k actin mRNA samples and cells Res. 2002, 62: 2546 antigen) Q01 overexpressed (partial in ~40% sequence) Pca; correlates with higher grade (Non-polarized distribution) STEAP Abnova Predominantly in Overexpressed in Prostate WO05113601A2 None None 1 (Six- Corporation: prostate; some prostate cancer epithelial (V_(H) & V_(L)) trans- H00026872- presence in (98%-positive in cells anti-STEAP-1 membrane P01 bladder; low level Pca, 97% epithelial (full length) in colon, positive in BPH) antigen of pancrease, the stomach, and prostate) uterus EphA2 R&D No normal Overexpressed in Prostate Methods. 2005, None None (Ephrin Systems: prostate IHC ~93% of prostate cancer cells 36(1): 43 receptor 3035-A2-100 staining cance samples by (V_(H) & V_(L)) A2) IHC (diffused into Mol. Immunol cytoplasm) 2007, 44: 3049 (EA2 & 47: V_(H) & V_(L)) EpCAM R&D Expressed on the Highly Epithelial Cancer Immunol IL2 fusion: β-glucuronidase (Epithelial Systems: baso-lateral cell upregulated in cells and Immunother. J Immunother. fusion: Br J cell 960-EP-050 surface in most ovarian cancer, prostate 2001, 50(1): 51. 2004, 27(3): 211 Cancer. 2002, adhesion human simple breast cancer, cancer cells Cancer Res. 86(5): 811 molecule) epithelia, very etc; increased in 1999 low exoression prostate cancer 59(22): 5758 in normal ovaries (V_(H) & V_(L)) ALCAM R&D Broad distribution, Strong cell surface Epithelial Reported in J. None Saporin S6 (Activated Systems: in epithelia, expression in 31% cells and Cell Biol. 2005, conjugate: J. Cell leukocyte 656-AL neurons, lymphoid colorectal other normal 118(7): 1515 & Biol. 2005, cell and myeloid cells, carcinoma; mRNA cells, and Liu B., et al. J. 118(7): 1515 adhesion hematopoietic and overexpression in prostate Mol. Med. 2007, molecule, mesenchymal stem 86% prostate cancer cells but sequences CD166) cells carcinoma were not disclosed EGFR? R&D Kidneys, liver, Upregulated in EGFR+ Int J Cancer. Taxol conjugate: PEA fusion: Systems: intestine, bone, cancers of colon, cancer cells 1995, 60: 137 Bioconjug Chem. Int J Cancer. 2000, 1095-ER-002 etc. breast, pancreas, (V_(H) & V_(L)) 2003, 14(2): 302 86(2): 269. J Nucl Med. etc. Mutated to Jpn J Cancer Methotrexate GrB-TGFα fusion: 2006, 47(6): 1023 EGFRvIII in Res. 2000 conjugate: Mol Cell Death Differ. Pca. 91(10): 1035 Cancer Ther. 2006, 2006 13(4): 576. (vIII V_(H) & V_(L)) 5(1): 52 HER2? R&D Liver, kidneys, Upregulated in HER2+ Biochemistry Herceptin- PEA fusion: Systems: spleen, etc. cancers of colon, cancer cells 1994, 33: 5451 geldanamycin J Biol Chem. 1994, 1129-ER-050 Br J Pharmacol. breast, prostate, (dcFv V_(H) & V_(L)) conjugate: 269(28): 18327. 2004, 143(1): 99 etc. J Mol Biol. Cancer Res. 2004 Breast Cancer Res 1996, 255(1): 28 64(4): 1460 Treat. 2003, (V_(H) & V_(L)) 82(3): 155. GrB fusion: Cell Death Differ. 2006 13(4): 576. EGFR- See above Advantages of bispecific targeting: not EGFR+ or US20060099205 None Bivalent PEA fusion: HER2? limited by a single marker and higher HER2+ A1: Bispecific Br J Cancer. 1996, target density, neither is achievable by cancer cells single chain FVs 74(6): 853. natural protease system, e.g., uPA/uPAR (V_(H) & V_(L)) Int J Cancer. 1996, 65(4): 538. p-Glyco- Abnova Low expression Upregulated after Drug- MRK-16: Biol PEA conjugate: PEA fusion: protein Corporation: chemotherapy resistant Chem. 1999, J Urol. 1993, Int J Cancer. 2001, (MDR1 H00005243-Q01 cancer cells 274(39): 27371 149(1): 174 94(6): 864 gene (partial C219: J Biol product) sequence) Chem. 1997, 272(47): 29784 Antigen Target Normal Cancer Stem Antibody Antibody ScFc Pair Antigen Availability Distribution Cell Marker Sequences Immunotoxins Immunotoxins Targeting Cancer Causing Stem Cells [CD44]/ CD44 R&D Systems: Ubiquitously Metastatic cancer WO05049082A2 None None [EpCAM] & 3660-CD-050 expressed on cells, breast cancer (H90: V_(H) & V_(L)) [CD133]/ different cell stem cells, prostate Int. J. Cancer 1996, [EpCAM] surfaces stem cells, 68: 232 Etc. colorectal cancer (CD44v6 V_(H) & V_(L)) stem cells, Gyn. Oncol. 1997, 66: 209 pancreatic cancer (CD44v7v8 V_(H) & V_(L)) stem cells, and head & neck cancer stem cells EpCAM R&D Systems: Expressed on the Breast cancer stem Cancer Immunol IL2 fusion: β-glucuronidase (aka ESA, 960-EP-050 baso-lateral cell cells, colon cancer Immunother. 2001, J Immunother. fusion: Br J Ber-EP4, surface in most stem cells, 50(1): 51 2004, 27(3): 211 Cancer. 2002, B38.1, and human simple colorectal cancer Cancer Res. 1999 86(5): 811 CD326) epithelia stem cells, and 59(22): 5758 pancreatic cancer (V_(H) & V_(L)) stem cells CD133 Abnova Hematopoitic Colon cancer stem N/A None None (aka AC133 Corporation: stem cells cells, glioblastoma and H00008842-Q01 stem cells, prostate prominin-1) (partial sequence) cancer stem cells, and heptocellular carcinoma stem cells CD34 Prospec: Hematopoitic AML stem cells J. Immunonol. Methods None None Pro-292 stem cells 1997, 201: 223 (V_(H) & V_(L)) CD24 Abnova B cells, Pancreatic cancer N/A Ricin A conjugate: None Corporation: granulocytes stem cells Int J Cancer. 1996, H00000934-P01 66(4): 526 (full length) CXCR4 Abnova Widely Prostate stem cells U.S. Pat. No. 7,005,503 None None Corporation: expressed in H00007852-Q01 normal tissues (partial sequence) CD166 R&D Systems: Broad-distribution, Colorectal cancer Reported in J. Cell Biol. None Saporin S6 (ALCAM: 656-AL in epithelia, stem cells 2005, 118(7): 1515 & Liu B., conjugate: J. Cell Activated neurons, lymphoid et al. J. Mol. Med. Biol. 2005, leukocyte cell and myeloid cells, 2007, but sequences were 118(7): 1515 adhesion hematopoietic and not disclosed molecule) mesenchymal stem cells p-Glyco- Abnova Low expression Higher expression MRK-16: Biol Chem. PEA conjugate: PEA fusion: protein Corporation: in stem cells 1999, 274(39): 27371 J Urol. 1993, Int J Cancer. 2001, (MDR1 H00005243-Q01 C219: J Biol Chem. 149(1): 174 94(6): 864 gene (partial sequence) 1997, 272(47): 29784 product)

B. Cell Targeting Moieties

The invention features protoxin fusion proteins and protoxin activator fusion proteins each containing a cell-targeting moiety. Such cell targeting moieties of the invention include proteins derived from antibodies, antibody mimetics, ligands specific for certain receptors expressed on a target cell surface, carbohydrates, and peptides that specifically bind cell surface molecules.

One embodiment of the cell-targeting moiety is a protein that can specifically recognize a target on the cell surface. The most common form of target recognition by proteins is antibodies. One embodiment employs intact antibodies in all isotypes, such as IgG, IgD, IgM, IgA, and IgE. Alternatively, the cell-targeting moiety can be a fragment or reengineered version of a full length antibody such as Fabs, Fab′, Fab2, or scFv fragments (Huston, et al. 1991. Methods Enzymol. 203:46-88, Huston, et al. 1988. Proc Natl Acad Sci USA. 85:5879-83). In one embodiment the binding antibody is of human, murine, goat, rat, rabbit, or camel antibody origin. In another embodiment the binding antibody is a humanized version of animal antibodies in which the CDR regions have grafted onto a human antibody framework (Queen and Harold. 1996. U.S. Pat. No. 5,530,101). Human antibodies to human epitopes can be isolated from transgenic mice bearing human antibodies as well as from phage display libraries based on human antibodies (Kellermann and Green. 2002. Curr Opin Biotechnol. 13:593-7, Mendez, et al. 1997. Nat Genet. 15:146-56, Knappik, et al. 2000. J Mol Biol. 296:57-86). The binding moiety may also be molecules from the immune system that are structurally related to antibodies such as reengineered T-cell receptors, single chain T-cell receptors, CTLA-4, monomeric Vh or Vl domains (nanobodies), and camelized antibodies (Berry and Davies. 1992. J Chromatogr. 597:239-45, Martin, et al. 1997. Protein Eng. 10:607-14, Tanha, et al. 2001. J Biol Chem. 276:24774-80, Nuttall, et al. 1999. Proteins. 36:217-27). A further embodiment may contain diabodies which are genetic fusions of two single chain variable fragments that have specificity for two distinct epitopes on the same cell. As an example, a diabody with an anti-CD19 and anti-CD22 scFv can be fused to a protoxin or protoxin activator in order to increase the affinity to B-cell targets (Kipriyanov. 2003. Methods Mol Biol. 207:323-33).

In another embodiment the cell-targeting moiety can also be diversified proteins that act as antibody mimetics. Diversified proteins have portions of their native sequence replaced by sequences that can bind to heterologous targets. Diversified proteins may be superior to antibodies in terms of stability, production, and size. One example is fibronectin type III domain, which has been used previously to isolate affinity reagents to various targets (Lipovsek and Pluckthun. 2004. J Immunol Methods. 290:51-67, Lipovsek, et al. 2007. J Mol Biol. 368:1024-41, Lipovsek, Wagner, and Kuimelis. 2004. U.S. Patent 20050038229). Lipocalins have been used for molecular diversification and selection (Skerra et al. 2005. U.S. Patent 20060058510). Lipocalins are a class of proteins that bind to steroids and metabolites in the serum. Functional binders to CTLA4 and VEGF have been isolated using phage display techniques (Vogt and Skerra. 2004. Chembiochem. 5:191-9). C-type lectin domains, A-domains and ankyrin repeats provide frameworks that can be oligomerized in order to increase the binding surface of the scaffold (Mosavi, et al. 2004. Protein Sci. 13:1435-48). Other diversified proteins include but are not limited to human serum albumin, green fluorescent protein, PDZ domains, Kunitz domains, charybdotoxin, plant homeodomain, and β-lactamase. A comprehensive review of protein scaffolds is described in (Hosse, et al. 2006. Protein Sci. 15:14-27, Lipovsek. 2005.). Those skilled in the art understand that many diverse proteins or protein domains have the potential to be diversified and may be developed and used as affinity reagents, and these may serve as bell-binding moieties in the context of combinatorial targeting therapy.

In another embodiment, the cell-targeting moiety can be a naturally occurring ligand, adhesion molecule, or receptor for an epitope expressed on the cell surface. Compositions of the ligand may be a peptide, lectin, hormone, fatty acid, nucleic acid, or steroid. For example, human growth hormone could be used as a cell-targeting moiety for cells expressing human growth hormone receptor. Solubilized receptor ligands may also be used in cases in which the natural ligand is an integral membrane protein. Such solubilized integral membrane proteins are well-known in the art and are easily prepared by the formation of a functional fragment of a membrane protein by removing the transmembrane or membrane anchoring domains to afford a soluble active ligand; for example, soluble CD72 may be used as a ligand to localize engineered protoxins to CD5 containing cells. Another example is the binding of urokinase type plasminogen activator (uPA) to its receptor uPAR. It has been shown that the region of u-PA responsible for high affinity binding (K_(d)≈0.5 nM) to uPAR is entirely localized within the first 46 amino acids called N-terminal growth factor like domain (N-GFD) (Appella, et al. 1987. J Biol Chem. 262:4437-40). Avemers refer to multiple receptor binder domains that have been shuffled in order to increase the avidity and specificity to specific targets (Silverman, et al. 2005. Nat Biotechnol. 23:1556-61). These receptor binding domains and ligands may be genetically fused and produced as a contiguous polypeptide with the protoxin or protoxin activator or they can be isolated separately and then chemically or enzymatically attached. They may also be non-covalently associated with the protoxin or protoxin activator.

In a previously reported example, Denileukin difitox is a fusion protein of DT and human interleukin (IL)-2 (Fenton and Perry. 2005 Drugs 65:2405). Denileukin difitox targets any cells that express IL-2 receptor (IL2R), including the intended target CTCL cells. Acute hypersensitivity-type reactions, vascular leak syndrome, and loss of visual acuity have been reported as side effects. Because human normal non-hematopoietic cells of mesenchymal and neuroectodermal origin may express functional IL2R, some cytotoxic effects observed could be due to a direct interaction between IL-2 and non-hematopoietic tissues. In order to overcome this toxicity, the invention features, for example, addition of a T cell marker as a second targeting element, e.g., CD3.

If the moiety is a carbohydrate such as mannose, mannose 6-phosphate, galactose, N-acetylglucosamine, or sialyl-Lewis X, it can target the mannose receptor, mannose 6-phosphate receptor, asialoglycoprotein receptor, N-acetylglucosamine receptor, or E-selectin, respectively. If the moiety comprises a sialyl-Lewis X glycan operably linked to a tyrosine sulfated peptide or a sulfated carbohydrate it can target the P-selectin or L-selectin, respectively.

As another example, the binding partners may be from known interactions between different organisms, as in a pathogen host interaction. The C-terminal domain of the Clostridium perfringens enterotoxin (C-CPE) binds with high affinity and specificity to the mammalian claudin3/4 adhesion molecules. Although claudins are components of most cells tight junctions, they are not typically exposed on the apical surface. The C-CPE can be appended to the protoxin or activator in order to localize one of the components of the combinatorial targeting to cells overexpressing unengaged claudin3/4, a condition of many types of cancers (Takahashi, et al. 2005. J Control Release. 108:56-62, Ebihara, et al. 2006. J Pharmacol Exp Ther. 316:255-60).

An example of a peptide moiety is the use of angiotensin to localize complexes to cells expressing angiotensin receptor. In another embodiment, the binding peptide could be an unnatural peptide selected from a random sequence library. One group has identified a peptide using phage display, termed YSA, which can specifically recognize EphA2 receptors. EphA2 is overexpressed in many breast cancers (Koolpe, et al. 2005. J Biol Chem. 280:17301-11, Koolpe, et al. 2002. J Biol Chem. 277:46974-9). In order to increase binding affinity, peptides may be multimerized through sequential repeated fusions or attachment to a dendrimer which can then be attached to the protoxin or protoxin activator.

In another embodiment, the cell-targeting moiety can be a nucleic acid that consists of DNA, RNA, PNA or other analogs thereof. Nucleic acid aptamers have been identified to many protein targets and bind with very high affinity through a process of in vitro evolution (Gold. 1991. U.S. Pat. No. 5,475,096, Wilson and Szostak. 1999. Annu Rev Biochem. 68:611-47). RNA aptamers specific for PSMA were shown to specifically localized conjugated gelonin toxin to cells overexpressing PSMA (Chu, et al. 2006. Cancer Res. 66:5989-92). The nucleic acid can be chemically synthesized or biochemically transcribed and then modified to include an attachment group for conjugation to the reengineered toxin. The nucleic acid may be directly conjugated using common crosslinking reagents or enzymatically coupled by processes known in the art. The nucleic acid can also be non-covalently associated with the protoxin.

The cell-targeting moiety may be identified using a number of techniques described in the art. Typically natural hormones and peptide ligands can be identified through reported interactions in the reported literature. Additionally, antibody mimics and nucleic acid aptamers can be identified using selection technologies that can isolate rare binding molecules toward epitopes of interest, such as those expressed on cancer cells or other diseased states. These techniques include SELEX, phage display, bacterial display, yeast display, mRNA display, in vivo complementation, yeast two-hybrid system, and ribosome display (Roberts and Szostak. 1997. Proc Natl Acad Sci USA. 94:12297-302, Boder and Wittrup. 1997. Nat Biotechnol. 15:553-7, Ellington and Szostak. 1990. Nature. 346:818-22, Tuerk and MacDougal-Waugh. 1993. Gene. 137:33-9, Gyuris, et al. 1993. Cell. 75:791-803, Fields and Song. 1989. Nature. 340:245-6, Mattheakis, et al. 1994. Proc Natl Acad Sci USA. 91:9022-6). Antibodies can be generated using the aforementioned techniques or in a traditional fashion through immunizing animals and isolating the resultant antibodies or creating monoclonal antibodies from plasma cells.

The targets of the cell-targeting moieties may be protein receptors, carbohydrates, or lipids on or around the cell surface. Examples of polypeptide modifications known in the art that may advantageously comprise elements of a cell surface target include glycosylation, sulfation, phosphorylation, ADP-ribosylation, and ubiquitination. Examples of carbohydrate modifications that may be distinctive for a specific lineage of cells include sulfation, acetylation, dehydrogenation and dehydration. Examples of lipid modification include glycan substitution and sulfation. Examples of lipids that may be distinctive for a specific targeted cell include sphingolipids and their derivatives, such as gangliosides, globosides, ceramides and sulfatides, or lipid anchor moieties, such as the glycosyl phosphatidyl inositol-linked protein anchor.

The cell-targeting moiety may indirectly bind to the target cell through another binding intermediary that directly binds to a cell surface epitope, as long as the cell-targeting moiety acts to localize the reengineered toxin to the cell surface. The targets of these binding modules may be resident proteins, receptors, carbohydrates, lipids, cholesterol, and other modifications to the target cell surface. The cell-targeting moiety can be joined to the protoxin either through direct translational fusions if the DNA encoding both species is joined. Alternatively, chemical coupling methods and enzymatic crosslinking can also join the two components. The cell-targeting moiety may contain sequences not involved in the structure or binding of the agent, but involved with other processes such as attachment or interaction with the protoxin.

Disclosed herein are cell-targeting moieties that act to localize modified toxins to the surface of target cells. In one embodiment, the cell-targeting moiety is one or more single-chain variable fragment (scFv) that specifically recognize epitopes on cells of patients with B-CLL. In another embodiment the cell-targeting moiety is one or more single-chain variable fragments (scFv) that specifically recognize CD5. In yet another embodiment the cell-targeting moiety is a single-chain variable fragment (scFv) that specifically recognizes B-cell markers CD19 and CD22. In one embodiment the scFv fragment includes one or more specific tag sequence (LPETG (SEQ ID NO:38)) that is used for enzymatic crosslinking induced by SortaseA. The tag sequence may be at the N-terminus, C-terminus, or at an internal position. In another embodiment the LPETG (SEQ ID NO:38) tag sequence is located near or at the C-terminus. The expression and functional reproduction of scFv is well-known in the art. The scFvs were produced through the expression in the E. coli periplasm and refolded in vitro using reported procedures for obtaining functional scFvs.

Described herein are examples of using known natural receptor ligands as cell-targeting moieties. Specifically the N-terminal domain of u-PA was fused directly to a protoxin in order to specifically target u-PAR. Also, a toxin based on the fusion between the C-terminal domain of the Clostridium perfringens enterotoxin (C-CPE) and toxins are also described herein that can target claudin3/4.

II. Protoxins

The protoxins of the invention are designed to be independently targeted to one or more preselected cell surface targets. In order to become active, the protoxin of the invention must be modified by a corresponding protoxin activator. In one embodiment, the invention features a protoxin containing a cytotoxic domain of one toxin and a translocation domain of the same or another toxin, and an intervening peptide containing a proteolytic cleavage sequence specifically recognized by an exogenous protease. Alternatively, or additionally, the toxin activity may be blocked by a chemical or peptide moiety. In these cases, the toxin will only become active when this chemical or peptide moiety is modified by either an exogenous enzyme (i.e., a protoxin activator) or by an activator natively present at or around the target cell. The toxin or protoxin fusion can be derived from any toxin known in the art, including, without limitation, Diphtheria toxin, Pseudomonas exotoxin A, Shiga toxin, and Shiga-like toxin, anthrax toxin, pore-forming toxins or protoxins such as proaerolysin, hemolysins, pneumolysin, Cryl toxins, Vibrio pro-cytolysin, or listeriolysin; Cholera toxin, Clostridium septicum alpha-toxin, Clostridial neurotoxins including tetanus toxin and botulinum toxin; gelonin; nucleic acid modifying agents such as pierisin-1, and ribosome-inactivating proteins (RIPs) such as Ricin, Abrin, and Modeccin.

A. Proteolytic Toxins

Because many proteases play an essential role in targeted cell death in vivo, they may be used as the toxin moiety for the present invention. For example, granzymes are exogenous serine proteases that are released by cytoplasmic granules within cytotoxic T cells and natural killer cells, and can induce apoptosis within virus-infected cells, thus destroying them; caspases are cysteine proteases that play a central role in the initiation and execution phases of apoptosis; and a proteolytic cascade during complement activation results in complement-mediated inflammation, leukocyte migration, and phagocytosis of complement-opsonized particles and cells, which eventually leads to a direct lysis of target cells and microorganisms as a consequence of membrane-penetrating lesions.

Most proteases involved in apoptosis or complement activation exist in the form of a zymogen until activated. Zymogens are proenzymes that are inhibited by a propeptide component within its own sequence, usually located at the N-terminus. One embodiment of the present invention utilizes such a proteolytic zymogen as the protoxin moiety, and a second proteolytic activity acting as an activator of the zymogen. Both the protoxin and protease fusions comprise a cell-targeting domain, and optionally a translocation domain to assist endocytosis. Examples of the cleavage site within the first zymogen and the protease within the activator fusion include, but are not limited to, a protease cleavage site targeted by Factor Xa, IEGR↓; and a protease cleavage site targeted by Enterokinase, DDDDK↓ (SEQ ID NO:25). Additional examples include granzymes, caspases, elastase, kallikreins, the matrix metalloprotease (MMP) family, the plasminogen activator family, as well as fibroblast activation protein.

Granzymes

U.S. Pat. No. 7,101,977 discloses that a chimeric protein comprising an apoptosis-inducing factor such as granzyme B and a cell-specific targeting moiety can induce cell death. GrB induces cell death by cleaving caspases (especially caspase-3), which in turn activates caspase-activated DNase. This enzyme degrades DNA, irreversibly inactivating the apoptotic cell. GrB also cleaves the protein Bid, which recruits the protein Bax and Bak to change the membrane permeability of mitochondria, causing the release of cytochrome c (which activates caspase 9), Smac/Diablo and Omi/HtrA2 (which suppress the inhibitor of apoptosis proteins (IAPs)), among other proteins.

In a preferred embodiment of the present invention, an apoptosis-inducing granzyme (e.g., granzyme B) may be constructed as the cytotoxic part of a protoxin. For example, in constructing a GrB-based protoxin, a proteolytic substrate sequence may be placed in the immediate front of granzyme B sequence, resulting in a GrB fusion that is activatable by a protease fusion that can specifically cleave the proteolytic substrate sequence.

Caspases

There are two types of apoptotic caspases: initiator (apical) caspases and effector (executioner) caspases. Initiator caspases (e.g. caspase-2, -8, -9 and -10) cleave inactive pro-forms of effector caspases, thereby activating them. Effector caspases (e.g. caspase-3, -6, -7) in turn cleave other protein substrates within the cell resulting in the apoptotic process. In vivo the initiation of this cascade reaction is regulated by caspase inhibitors. The caspase cascade can be activated by Granzyme B, released by cytotoxic T lymphocytes, which activates caspase-3 and -7; by death receptors (like FAS, TRAIL receptors and TNF receptor) which activate caspase-8 and -10; and by the apoptosome, regulated by cytochrome c and the Bcl-2 family, which activates caspase-9.

Because caspases are critically involved in the later stages of apoptosis regardless of the initial stimulus of apoptosis, the invention features the direct use of these activities, particularly the effector caspases, to initiate an apoptotic cascade independent of upstream cellular events. For example, in constructing a caspase-6 based protoxin, a procaspase-6 is used. The procaspase-6 comprises the mature caspase-6 sequence, an inhibitory sequence, and a proteolytic substrate sequence placed in between. The procaspase fusion is selectively activated by a protease fusion that can specifically cleave the proteolytic substrate sequence.

Proteases of the Complement System

The complement system is a biochemical cascade that helps clear pathogens from an organism. The complement system includes of a number of small proteins found in the blood, which work together to kill target cells by disrupting the target cell's plasma membrane. Over 20 proteins and protein fragments make up the complement system, including serum proteins, serosal proteins, and cell membrane receptors. The complement system is not adaptable and does not change over the course of an individual's lifetime, and, as such, it belongs to the innate immune system. However, it can be recruited and brought into action by the adaptive immune system.

There are three distinct pathways of complement activation—the classical pathway, the lectin pathway, and the alternative pathway. Complement activation proceeds in a sequential fashion, through the proteolytic cleavage of a series of proteins, and leads to the generation of active products that mediate various biological activities through their interaction with specific cellular receptors and other serum proteins. During the course of this cascade, a number of biological processes are initiated by the various complement components, which eventually lead to direct lysis of target cells. C1-C9 and factors B and D are the reacting components of the complement system. One preferred embodiment of the present invention involves the use of a protease involved in the complement activation cascade (e.g., proteolytic component of the C1-C9 and Factors B and D, preferably C3) as the toxin moiety within the protoxin fusion.

B. Bacterial Toxins

Examples of bacterial toxins that may be used in the protoxin fusion proteins of the invention are set forth below.

Pore Forming Toxins

In another aspect, the invention features a protoxin fusion protein containing a pore-forming toxin domain. These toxins bind to cellular membranes and upon an activation trigger, create channels (pores) in which essential ions and metabolites may diffuse. Representative pore-forming toxins that require modification to become active include but are not limited to Aeromonas hydrophila aerolysin, Clostridium perfringens ε-toxin, Clostridium septicum α-toxin, Escherichia coli prohaemolysin, hemolysins of Vibrio cholerae, and B. pertussis AC toxin (CyaA).

In the reengineered activatable pore-forming toxins “RAPFTs” of the invention, the trigger to convert the toxin from an inactive form to an active form can be altered from the native mechanism to an alternative mechanism. A preferred manner of alteration is to replace a native proteolytic activation site with an heterologous proteolytic site that is not normally operationally resident on the target cell. The heterologous proteolytic site may be added to or replace the original activation site, while either mutating or preserving the original residues as long as the endogenous activation does not occur prior to activation by the exogenous protease. Alternative sequences or chemical compositions that may be used in the RAPFT include substrates for proteases from the activating moiety other than those previously reported. These alternative substrates may be used as the modified proteolytic site in the RAPFT.

Other modifications to the activation site include but are not limited to phosphorylation, glycosylation, lipoylation, biotinylation, acetylation, ubiquitination, sumoylation, and esterification. These modifications must be paired with activating groups that can reverse, remove, or further alter these modifications in order to switch the RAPFT from the inactive to the active state or to a natively activatable state when used in a therapeutic context. In another embodiment, RAPFTs can possess a modification to a vital portion of the toxin other than the native activation site that inhibits pore formation unless that modification is reversed. An example of this would be phosphorylation of a residue in the hydrophobic loop that forms part of the pore and which interferes with native pore-forming activity. Only when the phosphate group is removed, for example, with a phosphatase, can the protoxin form functional pores.

The RAPFTs can also contain an optionally substituted cell targeting moiety described herein in addition to the native targeting domain as long as the substituted cell-targeting moiety operably replaces the localizing function of the targeting domain. Additionally, the native targeting domain can be eliminated or replaced partially or entirely by an optionally substituted cell-targeting moiety. Those skilled in the art understand methods to make deletions, insertions, site-directed mutations, and random mutations to the native pore-forming toxin within the encoding DNA sequences that are then represented as changes in the encoded amino acid sequences using established molecular cloning techniques. Optionally substituted cell-targeting moieties can be appended to the protoxin as a direct genetic fusion, or can be added through chemical or enzymatic crosslinking. The cell-targeting moieties may also be non-covalently associated with the protoxin through hydrophobic, metal binding, and other affinity-based interactions. Additional variants of cell-targeting moieties are described herein.

Other modifications of RAPFT include single amino acid substitutions or combinations of multiple substitutions that may aid in the synthesis of functional immunotoxins as well as modify the properties of the reengineered protein, such as solubility, immunogenicity, or pharmacokinetics (Sambrook J. 2001. Cold-Spring Harbor Press., Ausubel F. 1997 and updates. Wiley and Sons.).

Modifications can include the addition of purification tags for the purpose of preparation of the RAPFT. The protoxin can be modified to include modifiable amino acids such as cysteines and lysines in specific positions in the toxin. Modifying groups such as binding or inhibitory domains can be added to these amino acids through alkylation of the sulfhydryl or epsilon amino group. Mutations that affect the natural activity of the RAPFT can be introduced. For example, mutations such as C159S and W324A can be made that disrupt the GPI-binding site within the aerolysin pore-forming toxin. These mutations would reduce the non-specific binding of the reengineered toxin (MacKenzie, et al. 1999. J Biol Chem. 274:22604-9).

In one embodiment, the RAPFT may encode sequences that allow for posttranslational modifications in vivo or in vitro. These post translational modifications include but are not limited to protease cleavage sites, lipoylation signals, phosphorylation, glycosylation, ubiquitination, sumoylation sites, and a BirA biotinylation target sequences for the addition of biotin. The biotinylation can occur during protein synthesis within the host organism or afterwards in an in vitro reaction. Streptavidin-biotin interactions can be used to couple the pore-forming function with other desired functionalities.

In another embodiment, an artificial inhibitory region may be substituted for a natural inhibitory sequence. In the case of aerolysin, residues between 433-470 may be replaced with an alternative sequence or chemical moiety that exhibits an analogous regulatory role. This region may be an alternative polypeptide sequence or small molecule, carbohydrate, lipid, or nucleic acid modification. Only when this non-native region is removed or inactivated will the toxin be activated or converted to a form that can be easily activated by the target cell. For example, an inhibitory peptide that is distinct in its primary sequence can be attached to the native inhibitory pro-peptide, and pore-forming activity can be restored upon removal of said inhibitory pro-peptide.

In another embodiment, the functioning portions of the RAPFT (e.g., the binding domain, pore-forming domain, and inhibitory pro-region) are linked together through non-peptide bonds. These domains are may be connected covalently using disulfide bonds, chemically crosslinked with bireactive alkylating reagents, or enzymatically through the conjugation with SortaseA or transglutaminase (Parthasarathy, et al. 2007. Bioconjug Chem. 18:469-76, Tanaka, et al. 2004. Bioconjug Chem. 15:491-7). Alternatively, a pore-forming toxin may contain functioning portions that are non-covalently associated (e.g., hydrophobic interactions like leucine zippers or binding interactions like SH2 domain-phosphate interaction) in order to achieve a functioning complex of associated pore-forming agents.

Another embodiment features RAPFTs in which one or more amino acids are substituted with unnatural amino acids (e.g., f 4-fluorotryptophan in place of tryptophan (Bacher and Ellington. 2007. Methods Mol Biol. 352:23-34, Bacher and Ellington. 2001. J Bacteriol. 183:5414-25)).

The functional RAPFT, without limitation, may have one or more of the following modifications: single or multiple amino acid mutations, altered activation moieties, optionally substituted cell-targeting domains, non-native inhibitory pro-regions, and unnatural amino acids.

In one preferred embodiment the RAPFT is based on the aerolysin pore-forming toxin. Aerolysin is produced by the species Aeromonas and causes cytolysis in a non-cell-specific manner. The toxin is comprised of four distinct domains and the superstructure exists as a dimer in the non-membrane bound form (Parker, et al. 1994. Nature. 367:292-5). Once the toxin is localized to cell membrane, furin cleaves a target sequence between residues 427-432, a C-terminal pro-domain which inhibits pore formation when present (residues 433-470) is removed, and the toxin can oligomerize with other activated toxins on the surface of the same cell. A hydrophobic segment is then inserted across the lipid bilayer to create a channel between the extracellular domain and cytosol. In the wild type aerolysin toxin, Domain 1 contains an N-glycan binding domain that targets the natural toxin to cells, and domain 2 contains a glycosyl-phosphatidylinositol (GPI) binding domain. Domain 3 contains the pore-forming loop and Domain 4 contains the pro-domain, separated from the pore-forming section by a cleavable linker with a furin recognition site.

The invention features modifications of pore-forming toxins to make them more suitable for administration as part of a RAPFT. In one embodiment of the reengineered aerolysin toxin, Domain 1 which is the native N-glycan binding domain can be removed. In another embodiment, Domain 1 can be optionally substituted with a cell-targeting moiety, with or without removing Domain 1. If Domain 1 is not removed, the toxin may or may not contain mutations in the binding site that affect the affinity toward the target molecule on the cell surface. The cell-targeting moiety may be attached to the N-terminus, C-terminus, or to an internal residue, provided it does not interfere with pore-forming activity once the RAPFT is activated. The optionally substituted protoxin can be synthesized by a variety of methods described herein.

The present invention also features a modified aerolysin with the residues between the pore-forming section and the pro-domain that inhibits pore formation (residues 427-432) changed from the native protease cleavage site to a modifiable activation moiety. Some embodiments comprise a mutated activation moiety in which the native furin activation moiety is substituted by one or more alternative protease recognition sequences. The native furin cleavage sequence KVRR↓AR (SEQ ID NO:7) (residues 427-432) can be replaced with the granzyme B activation moiety (IEPD (SEQ ID NO:9)). In this case, the therapeutic regimen would pair this embodiment with a granzyme B moiety as the protoxin activator. Alternatively, the native furin sequence can be replaced by the tobacco etch virus protease (TEV). The different protease activation sites include but are not limited to those described herein. The DNA encoding the native activation moiety can be replaced with a modified sequence using standard molecular biology methods (Sambrook J. 2001. Cold-Spring Harbor Press. Ausubel F. 1997 and updates. Wiley and Sons.). Sequences that can be cleaved by exogenous proteases, but have not been yet identified as substrates, can also be used.

In another embodiment, the first 82 residues of aerolysin are removed through DNA mutagenesis. Here, the small lobe is replaced by a DNA encoded linker sequence in which a peptide sequence which can be recognized and modified by SortaseA is added (GKGGSNSAAS (SEQ ID NO:22)). A cell-binding moiety which has at its C-terminus a sortase A acceptor sequence (LPETG SEQ ID NO:38)) is coupled to the mutated toxin using immobilized sortaseA. Sortase A forms a covalent attachment between the C-terminus of the threonine from the single chain Fv and the N-terminus of the GKGGSNSAAS (SEQ ID NO:22). In a preferred embodiment the cell-binding moiety is a single chain Fv fragment. In another embodiment, the single chain Fv fragment has specificity towards the cell surface receptor CD5, which is normally found on T-cells and not B-cells. In the case of chronic B-cell chronic lymphoid leukemia (B-CLL), B-cells are found to have the receptor on the cell surface. In addition to this mutation, the reengineered aerolysin contains an alternative proteolytic activation site recognized by human Granzyme B in place of the native furin active (residues 427-432). When this reengineered aerolysin is paired with an activating moiety which has a granzyme B protease associated with a targeting module that also targets the diseased cell, as an example a granzyme B that has been functionally fused with a single-chain antibody fragment that can recognize CD19, a common B-cell marker, the reengineered aerolysin can become activated and destroy the cell expressing both CD5 and CD19 through the formation of a heptameric pore. In yet another embodiment the anti-CD5 and anti-CD19 moieties are swapped between the protoxin and protoxin activator. The aerolysin based RAPFT is modified with anti-CD19 and the the activating protease is modified with anti-CD5.

In another embodiment, the invention features RAPFTs based on homologous toxins to aerolysin such as Clostridium septicum alpha-toxin. This pore-forming toxin does not have a native N-glycan binding region, domain1, and thus can be modified to have a cell-targeting moiety apart from the GPI-binding domain. Analagous mutations to the activation region of alpha-toxin can be made as described for aerolysin.

Those skilled in the art understand how to express RAPFTs in a variety of host systems. In one embodiment the protoxin may be produced in the organism, or related organism from which the natural toxin is normally found. In order to simplify the production process reengineered toxins can also be produced in heterologous expression systems such as E. coli, yeast (e.g. Pichia pastoris, Kluvermyces lactis), insect cells, in vitro translation systems, and mammalian cells (eg. 293, 3T3, CHO, HeLa, Cos, BHK, MDCK) as described in standard molecular biology guides. Transcriptional regulators and translational signals can be incorporated within the commercially available vector systems that accompany the various heterologous expression systems. Expression of the protoxin can be targeted to the intracellular or extracellular compartments of the host cell through the manipulation of signal peptides. The reengineered toxins may be expressed in fragments in different expression systems or created synthetically and then subsequently reconstituted into functional RAPFTs using purified components.

PCT Application Publication No. WO 20071056867 teaches the use of modified pore-forming protein toxins (MPPTs). MPPTs are derived from naturally-occurring pore-forming protein toxins (nPPTs) such as aerolysin and aerolysin-related toxins, and comprise a modified activation moiety that permits activation of the MPPTs in a variety of different cancer types. WO 2007/056867 distinguishes MPPTs from the pore-forming molecules described in PCT Application Publication No. WO 03/1018611, which have been engineered to selectively target a specific type of cancer. The MPPTs of WO 2007/056867 are intended to be used as broad spectrum anti-cancer agents and accordingly are constructed to be activated by proteolytic enzymes found in a plurality of cancer types. The activation moieties of the present invention are cognate to exogenous proteases that are not native to the tumor or expected to be enriched in the vicinity of the tumor.

Bacterial Activatable ADP-Ribosylating Toxins (ADPRTs)

Several groups of bacterial ADPRTs are known to be proteolytically activated. Cholera toxin, pertussis toxin and the E. coli enterotoxin are members of the AB₅ family that target small regulatory G-proteins. The enzymatically active A subunit binds non-covalently to pentamers of B subunits (Zhang et al. J. Mol Biol. 251: 563-573 (1995)). Members of the AB5 family of ADP-ribosylating toxins, including pertussis toxin, E coli heat labile enterotoxin and cholera toxin, require that the catalytic domain (A) undergo proteolytic cleavage of the disulfide linked A1-A2 domain. Proteolytic cleavage of the A subunit results in the A1 domain being released from the A2-B5 complex, rendering the A2-B5 complex cytotoxic in the presence of a cellular cofactor (Holboum et al. FEBS J. 273:4579-4593 (2006))

Diphtheria toxin, Pseudomonas exotoxin, and Vibrio Cholera Exotoxin presented in the present invention are members of the AB family. AB family toxins are multi-domain proteins consisting of a cell targeting domain, a translocation domain and an ADRPT domain by which the toxin ADP ribosylates a diphthamide residue on eukaryotic elongation factor 2 (Hwang et al. Cell 48:229-236(1987); Collier. Bacteriol. Rev. 87:828-832(1980)).

The third group comprises the actin-targeting AB combinatorial toxins that, unlike the more common AB₅ combinatorial toxins, comprise two domains, an active catalytic domain and a cell-targeting domain. This group includes a wide range of clostridial toxins including C2 toxin from Clostridium botulinum, Clostridium perfringens Iota toxin, Clostridium spiroforme toxin, Clostridium difficile toxin and the vegetative insecticidal protein (VIP2) from Bacillus cereus (Aktories et al. Nature 322:390-392(1986); Stiles & Wilkins Infect and Immun 54: 683-688 (1986); Han et al. Nature Struct Biol 6:932-936 (1999)). Combinatorial toxins do not bind cells as complete A-B units. Instead proteolytically activated B monomers bind to cell surface receptors as homoheptamers. These homoheptamers then bind to the A domains and are taken into cells via endocytosis. Once inside acidic endosomes, the low pH activates the translocation function of the B domain heptamers and they translocate the catalytic A domains across the endosomal membrane into the cytoplasm where they ADP-ribosylate actin and cause cell death (Barth et al. Microbiol. Mol. Biol. Rev. 68:373-402 (2004))

ADP-ribosylating toxins of the present invention include those that can induce their own translocation across the target cell membranes, herein referred to as “autonomously acting ADP-ribosylating toxins,” which have no requirement for a type III secretion system or similar structure expressed by bacteria to convey the translocation of the toxin into the host cytoplasm by an injection pilus or related structure. Such autonomously acting ADP-ribosylating toxins can be modified with respect to their activation moiety and cell-targeting moiety and produced by methods well known in the art.

Like the autonomously acting ADP-ribosylating toxins from bacterial sources, the pierisin-1 toxin from the butterfly Pieris rapae can be activated by proteolytic cleavage at a trypsin-sensitive site, Arg-233; cleavage results in a nicked toxin that shows enhanced cytolytic activity and the fragment 1-233 is cytotoxic if electroporated into HeLa cells (Kanazawa et al. Proc Natl Acad Sci USA. 98(5):2226-31 (2001)). Arg-233 lies in a predicted disordered loop of sequence GGHRDQRSERSASS (SEQ ID NO:40) in which the third arginine residue is Arg-233. Pierisin-1 contains a C-terminal sphingolipid binding region that targets the toxin to eukaryotic membranes and is believed to consist of four repeats of a lectin-like domain similar to that found in the plant toxin ricin (Matsushima-Hibiya et al. J Biol Chem. Mar. 14, 2003; 278(II):9972-8). Mutation of tryptophan residues thought to comprise the carbohydrate-binding motif results in reduced activity of the toxin (Matsushima-Hibiya et al. J Biol Chem. Mar. 14, 2003; 278(11):9972-8). Hence the redirection of the toxin to novel cell surface targets can be achieved by addition of an exogenous cell-targeting moiety to an engineered variant of pierisin-1 or related toxin that lacks carbohydrate-binding capacity as a result of mutational modification to the coding sequence. Such redirected pierisin can be additionally modified in the activation moiety to replace the arginine-rich RDQRSER (SEQ ID NO:41) sequence with a sequence cognate to a protoxin-activating protease.

Another aspect of the present invention is the provision of a new protoxin moiety derived from Vibrio cholerae, hereinafter known as Vibrio cholerae exotoxin or VCE. Like the catalytic moieties of diphtheria toxin and Pseudomonas exotoxin A, the VCE catalytic moiety specifically ADP-ribosylates diphthamide on eEF2. ADP-ribosylation of diphthamide impairs the function of eEF2 and leads to inhibition of protein synthesis which results in profound physiological changes and ultimately cell death. The mechanism whereby VCE enters the cell is not fully understood, but the related toxin PEA binds to the α₂-macroglobulin receptor on the cell surface and undergoes receptor-mediated endocytosis, becoming internalized into endosomes where the low pH creates a conformational change in the toxin leaving it open to furin protease cleavage that removes the binding domain. The catalytic domain then undergoes retrograde transport to the endoplasmic reticulum, translocates into the cytoplasm and can enzymatically ribosylate eEF2. DT by contrast binds to the heparin binding epidermal growth factor-like growth factor precursor (HB-EGF) and is cleaved on the cell surface before uptake through receptor mediated endocytosis. Once in the early endosome, the DT catalytic fragment is not processed and penetrates the membrane of the endosome to pass directly into the host cell cytoplasm where it can ADP-ribosylate eEF2. The receptor responsible for binding of VCE is currently unknown. In several regards, VCE resembles PEA more closely than it resembles DT. First, the domain organization of VCE appears similar to that of PEA, in which the cell-targeting domain is followed by the translocation domain and then the enzymatic domain. VCE and PEA both possess a masked ER retention signal at the C-terminus, suggesting that VCE and PEA enter the cytosol of target cells via endoplasmic reticulum. Both VCE and PEA have low lysine content, thought to be consistent with the mechanism of introduction of toxin into the cytoplasm through the endoplasmic reticulum associated degradation (ERAD) pathway. The present data support the view that the proteolytic event that activates PEA and VCE occurs in an acidic endosomal compartment, whereas furin cleavage of DT might take place in a more neutral environment.

The C-terminus of VCE bears a characteristic endoplasmic reticulum retention signal (KDEL (SEQ ID NO:15)) followed by a lysine residue at the very C-terminus of the VCE which presumably will be removed by a ubiquitous carboxyl-peptidase activity such as carboxypeptidase B, suggesting that VCE enters the cytosol of target cell in a manner similar to PEA and that the C-terminal sequence of VCE is essential for full cytotoxicity. Thus, for maximum cytotoxic properties of a preferred VCE molecule, an appropriate carboxyl terminal sequence is preferred to translocate the molecule into the cytosol of target cells. Such preferred amino acid sequences include, without limitation, KDELK (SEQ ID NO:42), RDELK (SEQ ID NO:43), KDELR (SEQ ID NO:44) and RDELR (SEQ ID NO:45).

Generic methods similar to those described below for DT fusion proteins may be applied to prepare recombinant DNA constructs and to express modified VCE fusion proteins they encode. Specifically for VCE fusions, the cell-targeting moiety (residues 1-295) of wild type VCE is replaced by a polypeptide sequence that binds to a different, selected target cell surface target, and the furin cleavage sequence (residues 321-326: RKPR↓DL (SEQ ID NO:46)) is displaced by a recognition sequence of an exogenous protease such as GrB, GrM, and TEV protease.

In another embodiment the invention includes the use of modified Pseudomonas exotoxin A as an element of a protoxin. Many useful improvements of PEA are known in the art. For example deletion and substitution analyses have indicated that the C-terminus of PEA contains an element essential for the cytotoxic effect of PEA. Mutational analyses of the region between amino acid 602 and 613 identified the last 5 amino acid residues (RDELK (SEQ ID NO:43)) as essential for toxicity and a basic residue at 609 and acidic amino acid at 610, 611, and a leucine at 612 as required for full cytotoxicity, whereas the lysine at 613 was identified to be dispensable (Chaudhary et al. Proc. Natl. Acad. Sci. 87:308-312 (1990)). A mutant PEA in which the C-terminus RDELK (SEQ ID NO:43) sequence was replaced with KDEL (SEQ ID NO: 15), a well defined endoplasmic reticulum retention signal, is fully functional, suggesting that intoxication by PEA requires cellular factor(s) present in the target cells and that PEA protein might travel to the lumen of the endoplasmic reticulum. Subsequently, it was found that immunotoxins engineered to have a consensus endoplasmic reticulum retention signal at the C-termini exhibit higher toxicity that those with the wild type PEA sequences (Seetharam et al., J. Biol. Chem. 266:17376-17381 (1991); U.S. Pat. No. 5,705,163; U.S. Pat. No. 5,821,238). Hence one embodiment of the present invention includes modified PEA bearing C-terminal sequence changes that favorably improve the toxicity to tumor cells.

Generic methods similar to those described below for DT fusion proteins may be applied to prepare recombinant DNA constructs and to express modified PEA fusion proteins they encode. Specifically for PEA fusions, the cell-targeting moiety (residues 1-252) of wild type PEA is replaced by a polypeptide sequence that binds to a different, selected target cell surface target, and the furin cleavage sequence (residues 276-281: RQPR↓GW (SEQ ID NO:5)) is displaced by a recognition sequence of an exogenous protease such as GrB, GrM, and TEV protease.

Various modifications have been described in the art that improved toxicity of PEA. These modification are also useful for improving the toxicity of VCE immunotoxins. Mere et al. J. Biol. Chem. 280: 21194-21201 (2005) teach that exposure to low endosomal pH during internalization of Pseudomonas exotoxin A (PE) triggers membrane insertion of its translocation domain, a process that is a prerequisite for PEA translocation to the cytosol where it inactivates protein synthesis. Membrane insertion is promoted by exposure of a key tryptophan residue (Trp 305). At neutral pH, this residue is buried in a hydrophobic pocket closed by the smallest α-helix (helix F) of the translocation domain. Upon acidification, protonation of the Asp that is the N-cap residue of the helix leads to its destabilization, enabling Trp side chain insertion into the endosome membrane. A mutant PEA in which the first two N-terminal amino acids (Asp 358 and Glu 359) of helix F replaced with non-acidic amino acids, showed destabilization of helix F, leading to exposure of tryptophan 305 to the outside of the molecule in the absence of an acidic environment and resulting in 7-fold higher toxicity than wild type PEA. Similarly, the mutant PEA in which the entire helix F is removed was shown to exhibit 3-fold higher toxicity than wild type PEA. Hence one embodiment of the present invention includes modified PEA bearing sequence changes to helix F or Trp 305 that favorably improve the toxicity to tumor cells. Although by sequence alignment, we did not find a helix corresponding to the helix F of PE, we found that, similar to the proteolytic cleavage of PEA, cleavage of VCE by furin is favored in mildly acidic conditions, suggesting that a similar acid triggered conformational change might take place during membrane insertion of VCE. Mutations that facilitate membrane insertion of VCE, and thereby enhance cytotoxicity, might be found through means such as random mutagenesis. Thus, preferable forms of VCE molecules for the present invention include those that exhibit more efficient membrane insertion, leading to higher toxicity.

One of the important factors determining the toxicity of the PEA-based or VCE-based immunotoxins depends on whether the immunotoxins are internalized by the target cell upon receptor binding. The internalization is considered the rate-limiting step in immunotoxin-mediated cytotoxicity (Li and Ramakrishnan. J. Biol. Chem. 269: 2652-2659 (1994)). He et al. fused Arg₉-peptide, a well known membrane translocational signal, to an anti-CEA (carcinoembryonic antigen) immunotoxin, PE35/CEA(Fv)/KDEL, at the position between the toxin moiety and the binding moiety. Strong binding and internalization of this fusion protein was observed in all detected cell lines, but little cytotoxicity to the cells that lack the CEA molecules on the cell surface was detected. However, the cytotoxicity besides the binding activity of the fusion protein to specific tumor cells expressing large amount of CEA molecules on the cell surface was improved markedly, indicating that the Arg₉-peptide is capable of facilitating the receptor-mediated endocytosis of this immunotoxin, which leads to the increase of the specific cytotoxicity of this immunotoxin (He et al. International Journal of Biochemistry and Cell Biology, 37:192-205 (2005)). Accordingly, one preferred embodiment of protoxins that depend on translocation to the endoplasmic reticulum for intoxication includes the operable linkage of Arg9-peptide or related membrane translocation signals, such as, without limitation, those derived from HIV-Tat, Antennapedia, or Herpes simplex VP22, to such protoxins. A further preferred embodiment of the present invention includes modified PEA or VCE protoxins operably linked to Arg9-peptide or related membrane translocation signals, such as, without limitation, those derived from HIV-Tat, Antennapedia, or Herpes simplex VP22.

Toxicities that are independent of ligand binding have been observed with most targeted toxins. These include either hepatocyte injury causing abnormal liver function tests or vascular endothelial damage with resultant vascular leak syndrome (VLS). Both the hepatic lesion and the vascular lesion may relate to nonspecific uptake of targeted toxins by normal human tissues. U.S. Patent Application Publication No. 2006/0159708 A1 and U.S. Pat. No. 6,566,500 describe methods and compositions relating to modified variants of diphtheria toxin and immunotoxins in general that reduce binding to vascular endothelium or vascular endothelial cells, and therefore reduce the incidence of Vascular Leak Syndrome (VLS), wherein the (X)D(Y) sequence is GDL, GDS, GDV, IDL, IDS, IDV, LDL, LDS, and LDV. In one example, avariant of DT, V7AV29A, in which two (X)D(Y) motifs are mutated is shown to maintain full cytotoxicity, but to exhibit reduced binding activity to human vascular endothelial cells (HUVECs). U.S. Pat. No. 5,705,156 teaches the use of modified PEA molecules in which 4 amino acids (57, 246, 247, 249) in domain I are mutated to glutamine or glycine to reduce nonspecific toxicity of PEA to animals. Hence one embodiment of the present invention includes modified PEA, VCE, or DT protoxins bearing sequence changes that favorably reduce toxicity to normal tissues.

The plasma half-lives of several therapeutic proteins have been improved using a variety of techniques such as those described by Collen et al., Bollod 71:216-219 (1998); Hotchkiss et al., Thromb. Haemostas. 60:255-261 (1988); Browne wt al., J. Biol. Chem. 263:1599-1602 (1988); Abuchowski et al., Cancer Biochem. Biophys. 7:175 (1984)). Antibodies have been chemically conjugated to toxins to generate immunotoxins which have increased half-lives in serum as compared with unconjugated toxins and the increased half-life is attributed to the native antibody. WO94/04689 teaches the use of modified immunotoxins in which the immunotoxin is linked to the IgG constant region domain having the property of increasing the half-life of the protein in mammalian serum. The IgG constant region domain is CH2 or a fragment thereof. Similar strategy can be applied to creating variants of VCE immunotoxin with increased serum half-life. In addition operable linkage to albumin, polyethylene glycol, or related nonimmunogenic polymers may promote the plasma persistence of therapeutic toxins.

Upon repeated treatment of immunotoxins, patients may develop antibodies that neutralize, hence lessen the effectiveness of immunotoxins. To circumvent the problem of high titer antibodies to a given immunotoxin, U.S. Pat. No. 6,099,842 teaches the use of a combination of immunotoxins bearing the same targeting principle, but differing in their cytotoxic moieties. In one example, anti-Tac(Fv)-PE40 and DT(1-388)-anti-Tac(Fv) immunotoxins are used in combination to reduce the possibility of inducing human anti-toxin antibodies. A similar strategy may be applied to the present invention where the protoxins of a combinatory strategy can be alternated between two or more protoxins, for example, those described herein.

One particular type of toxin fusion protein, the DT fusion protein, can be produced from nucleic acid constructs encoding amino acid residues 1-389 of DT, in which the native furin cleavage site is replaced by a recognition sequence of an exogenous protease and a polypeptide that can bind to a cell surface target. Those skilled in the art will recognize a variety of methods to introduce mutations into the nucleic acid sequence encoding DT or to synthesize nucleic acid sequences that encode the mutant DT. Methods for making nucleic acid constructs are well known and well documented in publications such as Current Protocols in Molecular Biology (Ausubel et al., eds., 2005). The nucleic acid constructs can be generated using PCR. For example, the construct encoding the DT fusion protein can be produced by mutagenic PCR, where primers encoding an alternative protease recognition site can be used to substitute the DNA sequence coding the furin cleavage site RVRRSV (SEQ ID NO:47). Constructs containing the mutations can also be made through sequence assembly of oligonucleotides. Either approach can be used to introduce nucleic acid sequences encoding the granzyme B cleavage site IEPD (SEQ ID NO:9) in place of that which encodes RVRRSV (SEQ ID NO:47). In addition to IEPD (SEQ ID NO:9), GrB has been shown to recognize and cleave other similar peptide sequences with high efficiency, including IAPD (SEQ ID NO:48) and IETD (SEQ ID NO:49). These and other sequences specifically cleavable by GrB may be incorporated. Genetically modified proteases of higher than natural specificity or displaying a different specificity than the naturally occurring protease may be of use in avoiding undesirable side effects attributable to the normal action of the protease.

DNA sequences encoding a cell-targeting polypeptide can be similarly cloned using PCR, and the full-length construct encoding the DT fusion protein can be assembled by restriction digest of PCR products and the DT construct followed by ligation. The construct may be designed to position a nucleic acid sequence encoding the modified DT near the translation start site and the DNA sequence encoding the cell-targeting moiety close to the translation termination site. Such a sequence arrangement uses native Diphtheria toxin to confer optimal translocation efficiency of the catalytic domain of DT to the cytosol.

DT fusion proteins may be expressed in bacterial, insect, yeast, or mammalian cells, using established methods known to those skilled in the art, many of which are described in Current Protocols in Protein Science (Coligan et al., eds., 2006). DNA constructs intended for expression in each of these hosts may be modified to accommodate preferable codons for each host (Gustafsson et al., Trends Biotechnol. 22:346 (2004)), which may be achieved using established methods, for example, as described in Current Protocols in Molecular Biology (Ausubel et al., eds., 2005), e.g., site-directed mutagenesis. To quickly identify an appropriate host system for the production of a particular DT fusion, the Gateway cloning method (Invitrogen) may also be applied for shuffling a gene to be cloned among different expression vectors by in vitro site-specific recombination.

In addition to codon changes, other sequence modifications to the construct of a DT fusion protein may include naturally occurring variations of DT sequences that do not significantly affect its cytotoxicity and variants of the cell-targeting domain that do hot abolish its ability to selectively bind to targeted cells.

Further, the sequence of the cell-targeting domain can be modified to select for variants with improved characteristics, e.g., reduced immunogenicity, higher binding affinity and/or specificity, superior pharmacokinetic profile, or improved production of the DT fusion protein. Libraries of cell-targeting domains and/or DT fusions can be generated using site-directed mutagenesis, error-prone PCR, or PCR using degenerate oligonucleotide primers. Sequence modifications may be necessary to remove or add consensus glycosylation sites, for maintaining desirable protein function or introducing sites of glycosylation to reduce immunogenicity.

For high yield expression of DT fusion proteins, the encoding polynucleotide may be subcloned into one of many commercially available expression vectors, which typically contain a selectable marker, a controllable transcriptional promoter, and a transcription/translation terminator. In addition, signal peptides are often used to direct the localization of the expressed proteins, while other peptide sequences such as 6 His tags, FLAG tags, and myc tags may be introduced to facilitate detection, isolation, and purification of fusion proteins. To help successful folding of each domain within the DT fusion, a flexible linker may be inserted between the modified DT domain and the cell-targeting moiety in the expression construct.

DT fusion proteins may be expressed in the bacterial expression system Escherichia coli. In this system a ribosome-binding site is used to enhance translation initiation. To increase the likelihood of obtaining soluble protein fusion, its expression construct may include DNA that encodes a carrier protein such as MBP, GST, or thioredoxin, either 5′ or 3′ to the DT fusion, to assist protein folding. The carrier protein(s) may be proteolytically removed after expression. Proteolytic cleavage sites are routinely incorporated to remove protein or peptide tags and generate active fusion proteins. Most reports on successful E. coli expression of fusion proteins containing a DT moiety have been in the form of inclusion bodies, which may be refolded to afford soluble proteins.

DT fusion proteins may be expressed in the methylotrophic yeast expression system Pichia pastoris. The expression vectors for this purpose may contain several common features, including a promoter from the Pichia alcohol oxidase (AOX1) gene, transcription termination sequences derived from the native Pichia AOX1 gene, a selectable marker wild-type gene for histidinol dehydrogenase HIS4, and the 3′AOX1 sequence derived from a region of the native gene that lies 3′ to the transcription termination sequences, which is required for integration of vector sequence by gene replacement or gene insertion 3′ to the chromosomal AOX1 gene. Although P. pastoris has been used successfully to express a wide range of heterologous proteins as either intracellular or secreted proteins, secretion is more commonly used because Pichia secretes very low levels of native proteins. A secretion signal peptide MAT factor prepro peptide (MF-α1) is often used to direct the expressed protein to the secretory pathway.

Post-translational modification such as N-linked glycosylation in Pichia occurs by adding approximately 8-14 mannose residues per side chain. Although considered less antigenic than the extensive modifications in S. cerevisiae (50-150 mannose residues per side chain), there is still a possibility that such glycosylation could elicit immune responses in human. Therefore, any consensus N-glycosylation sites NXS(T) within an expression construct are typically mutated to avoid glycosylation.

DT is potently toxic to eukaryotic cells if the catalytic domain translocates to or is localized to the cytosol. Although Pichia is sensitive to diphtheria toxin, it has a tolerance to levels of DT that were observed to intoxicate other wild type eukaryotic cells and the expression of DT fusion by the secretory route has been successful (Woo et al., Protein Expr. Purif. 25:270 (2002)). Because the secretion of expressed heterologous protein in Pichia involves cleavage of signal peptide MF-α1 by Kex2, a furin-like protease, a DT fusion protein with its furin cleavage site replaced should be less toxic to Pichia than wild type DT fusion proteins. Alternatively, DT fusion proteins can be expressed in a mutant strain of Pichia, whose chromosomal EF-2 locus has been mutated to resist GDP ribosylation by catalytic domain of DT (Liu et al., Protein Expr. Purif. 30:262 (2003)).

DT fusion proteins may also be expressed in mammalian cells. Mutant cell lines that confer resistance to ADP-ribosylation have been described (Kohno and Uchida, J. Biol. Chem. 262:12298 (1987); Liu et al., Protein Expr. Purif. 19:304 (2000); Shulga-Morskoy and Rich, Protein Eng. Des. Sel. 18:25 (2005)) and can be used to express soluble DT fusion proteins. For example, a toxin-resistant cell line CHO—K1 RE1.22c has been selected and used to express a DT-ScFv fusion protein (Liu et al., Protein Expr. Purif. 19:304 (2000)) and a mutant 293T cell line has been selected and used to express a DT-IL7 fusion protein (Shulga-Morskoy and Rich, Protein Eng. Des. Sel. 18:25 (2005)). It has been determined that a G-to-A transition in the first position of codon 717 of the EP-2 gene results in substitution of arginine for glycine and prevents post-translational modification of diphthamide at histidine 715 of EF-2, which is the target amino acid for ADP-ribosylation by DT. EF-2 produced by the mutant gene is fully functional in protein synthesis (Foley et al., Somat. Cell Mol. Genet. 18:227 (1992)). Based on this information and established methods such as described in Current Protocols in Molecular Biology (Ausubel et al., eds., 2005), different mammalian cells may be transfected with vectors containing G717A mutant of EF-2 gene and select for cells that are resistant to DT.

Stable expression in mammalian cells also requires the transfer of the foreign DNA encoding the fusion protein and transcription signals into the chromosomal DNA of the host cell. A variety of vectors are commercially available, which typically contain phenotypic markers for selection in E. coli (Ap^(r)) and CHO cells (DHFR), a replication origin for E. coli, a polyadenylation sequence from SV40, a eukaryotic origin of replication such as SV40, and promoter and enhancer sequences. Based on methods described in Current Protocols in Protein Science (Coligan et al., eds., 2006), and starting with the DT-resistant cell lines, vectors containing DNA encoding DT fusion proteins may be used to transfect host cells, which may be screened for high producers of the fusion proteins.

Although mammalian expression systems are often used to take advantage of its post-translational modifications that are innocuous to human, this is not necessarily applicable to DT fusion proteins involved in the present invention. Because DT is of bacterial origin, potential N-glycosylation sites within its sequence may need to be mutated in order to retain the cytotoxicity potential of native DT. Further, glycosylation within cell-targeting domain may need to be avoided to maintain its desirable binding characteristics. However, consensus N-glycosylation sites may be introduced to linkers or terminal sequences so that such glycosylation do not hamper the functions of DT and cell-targeting moiety.

Proteinaceous Toxins

A common property of many proteinaceous toxins that might be deployed as therapeutic agents is their requirement for activation by proteolytic cleavage through the action of ubiquitous proteases such as furin/kexin proteases found in, on, or in the vicinity of, the target cell. One promising approach to increase the selectivity of highly active proteinaceous toxins has been the introduction of proteolytic cleavage sites to replace the endogenous recognition sequence with that of proteases hypothesized or known to be enriched in the tumor. For example a variant anthrax toxin has been engineered to replace the endogenous furin cleavage site with a site easily cleaved by urokinase, a protease often highly expressed by malignant cells (Liu et al. J Biol Chem. May 25, 2001; 276(21):17976-84.) The formation of a chimeric toxin consisting of anthrax lethal factor fused to the ADP-ribosylation domain of Pseudomonas exotoxin A resulted in an agent that selectively killed tumor cells (Liu et al. J Biol Chem. May 25, 2001; 276(21):17976-84.) The recombinant toxin in this case was natively targeted, i.e. did not comprise an independent tumor-specific targeting moiety. A recombinant anthrax toxin variant activatable by urokinase has been disclosed that may have broad applicability to various human solid tumors (Abi-Habib et al., Mol Cancer Ther. 5(10):2556-62 (2006)) Singh et al. Anticancer Drugs. 18(7):809-16 (2007) disclose the creation of recombinant aerolysins that can be activated by the chymotrypsin-like protease, prostate specific antigen.

Bacillus anthracis produces three proteins which when combined appropriately form two potent toxins, collectively designated anthrax toxin. Protective antigen (PA) and edema factor combine (EF) to form edema toxin (ET), while PA and lethal factor (LF) combine to form lethal toxin (LT) (Leppla et al. Academic Press, London 277-302 (1991)). A unique feature of these toxins is that LF and EF have no toxicity in the absence of PA, apparently because they cannot gain access to the cytosol of eukaryotic cells. PA is responsible for targeting of LT and ET to cells and is capable of binding to the surface of many types of cells. After PA binds to a specific receptor, it is cleaved at a single site by furin or furin-like proteases, to produce an amino-terminal 19 kD fragment that is released from the receptor/PA complex (Singh et al. J. Biol. Chem. 264:19103-19107 (1989)). Removal of this fragment from PA exposes a high affinity binding site for LF and EF on the receptor-bound 63 kD carboxyl-terminal fragment (PA63). The complex of PA63 and LF or EF enter cells and probably passes through acidified endosomes to reach the cytosol.

U.S. Pat. No. 5,677,274 teaches the use of modified PA in which the furin cleavage site is replaced with intracellular protease activatable sequences. Once cleaved by protease resident in target cells, cleaved PA presents a high affinity binding domain for a second fusion protein comprising a fragment of LF which binds to PA and a toxin moiety such as pseudomonas exotoxin which kills target cells. In one embodiment of the invention, the furin cleavage site was replaced with a HIV protease site, rendering the modified PA proteins to be activated specifically by HIV-infected cells or cells expressing HIV protease. Thus allows the fusion protein comprising a PA binding domain of LF and the translocation domain and ADPRT domain of PE to enter and kill target cells. In another embodiment, the furin cleavage sequence is replaced with an HIV cleavage sequence so that two proteolytic events are required to activate modified LF.

Anthrax lethal toxin, a protoxin of Bacillus anthracis, may also be employed according to the present invention. Anthrax lethal toxin has two components, a catalytic moiety that is a protease specific for mitogen-activated protein kinase kinases (MAPKK), and a cell-targeting and translocation moiety. The latter is referred to as protective antigen, and binds cells through widely distributed cell surface targets known as anthrax toxin receptors. Following activation by proteolytic cleavage at a furin-like recognition sequence, RKKR(SEQ ID NO:49), spanning residues 164 to 167 of the protective antigen, an inhibitory fragment is liberated and the remaining protective antigen fragment forms a heptamer that binds three catalytic moieties that are subsequently endocytosed. The activated protective antigen forms a pore in the acidic environment of the endosome, allowing the toxic catalytic moiety to enter the cell, where it causes the cleavage of mitogen activated protein kinase kinases, (MAPKKs), resulting in cell cycle arrest. Protective antigen can also bind anthrax edema factor and fusion proteins of lethal toxin and another toxin, such as PEA, have been exemplified in the art (Liu et al. J Biol Chem. 276(21):17976-84 (2001)).

Accordingly, replacement of the furin-like recognition sequence with that of an exogenous protease will result in a protoxin that is activatable by a second protoxin activating moiety. The protective antigen can be made to target specific cells through the replacement of the endogenous receptor binding domain with a cell target binding moiety that is selective for a target desirable for therapeutic purposes.

AB Toxins

A large class of bacterial toxins well-known in the art and particularly suitable for the purposes of this invention are known as AB toxins. AB toxins consist of a cell-targeting and translocation domain (B domain) as well as a enzymatically active domain (A domain) and undergo translocation into the cytoplasm following the action of an endogenous target cell protease on an activation sequence.

The AB toxins Bordetella dermonecrotic toxin (DNT), E. coli cytotoxic necrotizing factor 1 or 2 (CNF1 or CNF2) and Yersinia cytotoxic necrotizing factor (CNFY) may accordingly be used for the purposes of the present invention. These toxins are similar in structure and mechanism of action (Hoffmann and Schmidt, Rev Physiol Biochem Pharmacol. 152:49-63 (2004)). DNT is a transglutaminase that inactivates Rho GTPases by polyamination or deamidation (Schmidt et al. J Biol Chem. 274(45):31875-81 (1999); Fukui and Horiuchi, J Biochem (Tokyo). 136(4):415-9 (2004)). CNF1, CNF2 and CNFY are deamidases that deamidate Gln 63 or Rho GTPase (Schmidt et al., Nature 387(6634):725-9 (1997), Hoffmann and Schmidt, Rev Physiol Biochem Pharmacol. 152:49-63 (2004)). DNT comprises a membrane targeting domain at the N terminus known as the B domain, a furin-like protease cleavage site, a translocation domain, and a catalytic domain; to enter the cytoplasm DNT must bind its target cells, undergo internalization and cleavage, and be translocated across the membrane (Fukui and Horiuchi, J Biochem (Tokyo). 136(4):415-9 (2004)). According to the present invention, modified DNT can be provided in which the B domain is replaced by a heterologous cell-targeting moiety, or in which a heterologous cell-targeting moiety is added to an intact B domain, and the furin-like protease cleavage site is replaced with a modifiable activation sequence that may be modified by an exogenous activator. CNFY and CNF1 exhibit 61% sequence identity in a pattern of uniform divergence throughout the molecule. CNFY and CNF1 target the same residue of RhoA but use different cell surface receptors to enter the cell (Blumenthal et al. Infect Immun. 75(7):3344-53 (2007)). Entry appears to be through an acidified endosomal compartment (Blumenthal et al. Infect Immun. 75(7):3344-53 (2007)). According to the present invention, modified DNT, CNF1, CNF2, or CNFY can be provided in which the endogenous cell-targeting domain is replaced by a heterologous cell-targeting moiety, or in which a heterologous cell-targeting moiety is added to an intact endogenous cell-targeting domain, and the furin-like protease cleavage site is replaced with a modifiable activation sequence that may be modified by an exogenous activator.

Clostridial glucosylating cytotoxins may also be used for the purposes of the present invention. Toxins in this family transfer glucose or N-acetylglucosamine to Rho family GTPases following internalization and translocation of the toxin enzymatic moiety into the cytoplasm (Schirmer and Aktories, Biochim Biophys Acta. 1673(1-2):66-74 (2004)). Like AB toxins, the glucosylating cytotoxins undergo proteolytic cleavage to transfer the catalytic N-terminus into the host (Pfeiffer et al. J Biol Chem. 278(45):44535-41 (2003)).

Additional Modifications

In addition to the above, functional toxins may be generated through refolding insoluble toxins through rapid dilution or stepwise removal of denaturant in the presence of additives that prevent aggregation (Middelberg. 2002. Trends Biotechnol. 20:437-43).

Reengineered toxins may have encoded affinity tags from which one can use affinity chromatography methods to obtain purified samples. These tags can be used for purification and may also aid in the soluble expression of some embodiments. Examples include and are not limited to histidine tags, avidin/streptavidin interacting sequences, glutathione-S-transferase (GST), maltose-bining protein, thioredoxin, and FLAG encoding sequence tags. The protoxins may be purified from host cells by standard techniques known in the art, such as gel filtration, ion exchange, metal chelating, and affinity purification. The optionally substituted cell-targeting moiety may be attached to the pore-forming-agent through a linker that provides conformational freedom or spatial separation for the pore-forming agent to function properly. This linker can be a polypeptide and may be directly encoded on the DNA by means of a genetic fusion at the N or C-terminus, or at an internal position such as an exposed loop. The linker may possess specific features that will allow attachments to binding or regulatory moieties, such as target sequences for crosslinking enzymes such as transglutaminase or sortaseA (see conjugation methods). The linker may be synthetic such as a poly-ethylene glycol group or a long hydrocarbon chain and can be attached to the toxin (pore-forming agent) through chemical or enzymatic means such as alkylation or transglutaminase reaction. The linker need not be covalently associated with either the toxin or the cell-targeting moiety. The interactions can be through metal chelation, hydrophobic interactions, and small molecule protein interactions like biotin-streptavidin as long as the association does not interfere with the toxin upon activation.

C. Other Toxins

RIPs are enzymes that trigger the catalytic inactivation of ribosomes and other substrates. Such toxins are present in a large number of plants and have been found also in fungi, algae, and bacteria. RIPs are currently classified as belonging to one of two types: type 1, comprising a single polypeptide chain with enzymatic activity, and type 2, comprising two distinct polypeptide chains, an A chain equivalent to the polypeptide of a type 1 RIPs and a B chain with lectin activity. Type 2 RIPs known in the art may be represented by the formulae A-B, (A-B)₂, (A-B)₄ and or by polymeric forms comprising multiple B chains per A chain. Linkage of the A chain with B chain is through a disulfide bond. The toxic activity of RIPs is due to translational inhibition, a consequence of the hydrolysis of an N-glycosidic bond of a specific adenine base in a highly conserved loop region of the 28 S rRNA of the eukaryotic ribosome (Girbes et al, Mini Rev. Med. Chem. 4(5):461-76 (2004)).

RIPs are often initially produced in an inactive, precursor form. For example, ricin is initially produced as a single polynucleotide (preproricin) with a 35 amino acid N-terminal presequence and a 12 amino acid linker between the A and B chains. The presequence is removed during translocation of the ricin precursor into the endoplasmic reticulum. The protoxin is then translocated into specialized organelles called protein bodies where a plant protease cleaves at the linker region between A and B chains. U.S. Pat. No. 6,803,358 discloses a protoxin comprising ricin A chain, ricin B chain, and a heterologous protease-sensitive peptide linker that may be selectively activated by a tumor-associated protease (e.g., MMP-9) that cleaves the peptide linker.

The toxicity of RIPs to animals is highly variable, although type 1 RIP and the A-chains of type 2 RIP share the same catalytic activity. Although some type 1 RIPs are highly active in cell free translation systems, they may be much less toxic than the type 2 RIPs in vivo. This is thought to be due to the absence of the lectin chain, resulting in a low rate of penetration into cells. Among the toxic type 2 RIPs are some of the most potent toxins known, but the lethal doses of toxic type 2 RIP may also vary greatly among different toxins, as reported for abrin and ricin, modeccin, and volkensin (Battelli Mini Rev. Med. Chem. 4(5):513-21 (2004)).

One embodiment of the present invention uses a protoxin comprising a type 1 RIP or the A chain of type 2 RIP as toxin moiety, a cell-targeting moiety, and a linker containing an exogenous protease cleavage site linking the two moiety. This protoxin is used in conjunction with an activator, which comprises a protease that cleaves the heterologous protease cleavage site and a cell-targeting domain.

Another embodiment of the present invention is to use a protoxin comprising a type 1 or the A chain of type 2 RIP containing a presequence mutated to include an exogenous protease sensitive site and a cell-targeting moiety. This protoxin is used in conjunction with an activator, which comprises a protease that can cleave the heterologous protease cleavage site and a cell-targeting domain.

Examples of type 1 RIPs include, but not limited to bryodin, gelonin, momordin, PAP-S, saporin-S6, trichokirin and momorcochin-S. Examples of toxic type 2 RIP include, but not limited to Abrin, Modeccin, Ricin, Viscumin, and Volkensin.

Like the autonomously acting ADP-ribosylating toxins from bacterial sources, the pierisin-1 toxin from the butterfly Pieris rapae can be activated by proteolytic cleavage at a trypsin-sensitive site, Arg-233; cleavage results in a nicked toxin that shows enhanced cytolytic activity and the fragment 1-233 is cytotoxic if electroporated into HeLa cells (Kanazawa et al. Proc Natl Acad Sci USA. 98(5):2226-31 (2001)). Arg-233 lies in a predicted disordered loop of sequence GGHRDQRSERSASS (SEQ ID NO:40) in which the third arginine residue is Arg-233. Pierisin-1 contains a C-terminal sphingolipid binding region that targets the toxin to eukaryotic membranes and is believed to consist of four repeats of a lectin-like domain similar to that found in the plant toxin ricin (Matsushima-Hibiya et al. J Biol Chem. Mar. Mar. 14, 2003; 278(11):9972-8). Mutation of tryptophan residues thought to comprise the carbohydrate-binding motif results in reduced activity of the toxin (Matsushima-Hibiya et al. J Biol Chem. Mar. 14, 2003; 278(11):9972-8). Hence the redirection of the toxin to novel cell surface targets can be achieved by addition of an exogenous cell-targeting moiety to an engineered variant of pierisin-1 or related toxin that lacks carbohydrate-binding capacity as a result of mutational modification to the coding sequence. Such redirected pierisin can be additionally modified in the activation moiety to replace the arginine-rich RDQRSER (SEQ ID NO:41) sequence with a modifiable activation moiety that can be activated by an exogenous activator.

D. Toxin Modifications and Methods of Expressing Fusion Proteins

Expressing reengineered pore-forming toxins in a variety of host systems is well known in the art. In one embodiment the protoxin may be produced in the organism, or related organism from which the natural toxin is normally found. In order to simplify the production process reengineered toxins can also be produced in heterologous expression systems such as E. coli, yeast (e.g. Pichia pastoris, Kluvermyces lactis), insect cells, in vitro translation systems, and mammalian cells (eg. 293, 3T3, CHO, HeLa, Cos, BHK, MDCK) as described in standard molecular biology guides. Transcriptional regulators and translational signals can be incorporated within the commercially available vector systems that accompany the various heterologous expression systems. Expression of the toxin can be targeted to the intracellular or extracellular compartments of the host cell through the manipulation of signal peptides. The reengineered toxins may be expressed in fragments in different expression systems or created synthetically and then subsequently reconstituted into functional reengineered pore-forming toxins using purified components.

Due to the challenges of expressing large fusion proteins in soluble form, it may be advantageous to separately express different domains of these fusion proteins followed by chemical conjugation or enzymatic ligation. Either the toxin fusion or the protease fusion may be prepared using this strategy. For example, the cell-targeting moiety replacing the small lobe and the large lobe of aerolysin may be expressed in properly tagged subunits, which can then be crosslinked using various protein conjugation and ligation methods, including native chemical ligation (Yeo et al., Chem. Eur. J. 10:4664 (2004)), transglutaminase catalyzed ligation through the formation of a γ-glutamyl-ε-lysyl bond (Ota et al., Biopolymers 50(2):193 (1999)), and sortase-mediated ligation through a sequence specific transpeptidation (Mao et al., J. Am. Chem. Soc. 126:2670 (2004)).

In another embodiment, functional toxins may be generated through refolding insoluble toxins through rapid dilution or stepwise removal of denaturant in the presence of additives that prevent aggregation.

III. Protoxin Activator Fusion Protein Constructs

As described above, the invention features protoxin activator fusion proteins containing a cell targeting moiety and a modification domain. In a preferred embodiment, the modification domain includes the activity of an exogenous protease.

A. Exogenous Protease Selection

An exogenous protease and corresponding cleavage site may be chosen for the present invention based on the following considerations. The protease is preferably capable of cleaving a protoxin activation moiety without significantly inactivating the protoxin or itself. The protease is preferably not naturally found in or on cells that are desired to be spared, with the exception that the protease can be naturally found in such cells if its natural location does not allow it to activate an externally administered protoxin. For example, an intracellular protease such as a caspase may be used if the toxin must be activated at the surface of the cell or in some intracellular vesicular compartment that does not naturally contain the intracellular protease, such as the endosome, golgi, or endoplasmic reticulum. In such cases the cells that are desired to be spared could contain the protease but the protease would not activate the protoxin.

The catalytic activity of the protease Is preferably stable to in vivo conditions for the time required to exert its therapeutic effect in vivo. If the therapeutic program requires the repeat administration of the protease, the protease is preferably resistant to interference by the formation of antibodies that impair its function, for example neutralizing antibodies. In some embodiments the protease has low immunogenicity or can be optionally substituted to reduce immunogenicity or can be optionally substituted to reduce the effect of antibodies on its activity. The protease preferably has low toxicity itself or has low toxicity in the form of its operable linkage with one or more cell surface binding moieties. The protease is preferably stable or can be made to be stable to conditions associated with the manufacturing and distribution of therapeutic products. The protease is preferably a natural protease, a modified protease, or an artificial enzyme.

Desirable proteases of the present invention include those known to have highly specific substrate selectivities, either by virtue of an extended catalytic site or by the presence of specific substrate-recognition modules that endow a relatively nonselective protease with appropriate specificity. Proteases of limited selectivity can also be made more selective by genetic mutation or chemical modification of residues close to the substrate-binding pocket.

As is known in the art, many proteases recognize certain cleavage sites, and some specific, non-limiting examples are given below. One of skill in the art would understand that cleavage sites other than those listed are recognized by the listed proteases, and can be used as a general protease cleavage site according to the present invention.

Proteases of human origin are preferred embodiments of the present invention due to reduced risk of immunogenicity. A human protease utilizing any catalytic mechanism, i.e., the nature of the amino acid residue or cofactor at the active site that is involved in the hydrolysis of the peptides and proteins, including aspartic proteases, cysteine proteases, metalloproteases, serine proteases, and threonine proteases, may be useful for the present invention.

Because model studies of a potential therapeutic agent must be conducted in animals to determine such properties as toxicity, efficacy, and pharmacokinetics prior to clinical trials in human, the presence of proteinase inhibitors in the plasma of animals could also limit the development of therapeutics comprising proteolytic activities. The proteinase inhibitors in animal plasma can possess inhibitory properties that are different from their human counterparts. For example human GrB has been found to be inhibited by mouse serpina3n, which is secreted by cultured Sertoli cells and is the major component of serpina3 (α₁-antichymotrypsin) present in mouse plasma (Sipione et at., J. Immunol. 177:5051-5058 (2006)). However, the human α₁-antichymotrypsin has not been shown to be an inhibitor of human GrB. The difference between mouse and human plasma protease inhibitors may be traced to their genetic differences. Whereas the major human plasma protease inhibitors, α₁-antitrypsin and α₁-antichymotrypsin, are each encoded by a single gene, in the mouse they are represented by clusters of 5 and 14 genes, respectively. Even though there is a high degree of overall sequence similarity within these clusters of inhibitors, the reactive-center loop (RCL) domain, which determines target protease specificity, is markedly divergent. To overcome inhibition by mouse proteases, the screening and mutagenesis strategies described herein can be applied to identify mutant proteases that are resistant to inhibition by inhibitors present in the animal model of choice.

Human Granzymes

Recombinant human granzyme B (GrB) may be used as an exogenous protease within the protease fusion protein. GrB has high substrate sequence specificity with a consensus recognition sequence of IEPD and is known to cleave only a limited number of natural substrates. GrB is found in cytoplasmic granules of cytotoxic T-lymphocytes and natural killer cells, and thus should be useful for the present invention provided these cells are not the targeted cells. The optimum pH for GrB activity is around pH 8, but it retains its activity between pH 5.5 and pH 9.5 (Fynbo et al., Protein Expr. Purif. 39:209 (2005)). GrB cleaves peptides containing IEPD with high efficiency and specificity (Harris et al., J. Biol. Chem. 273:27364 (1998)). Because GrB is involved in regulating programmed cell death, it is tightly regulated in vivo. In addition, GrB is a single chain and single domain serine protease, which could contribute to a simpler composite structure of the fusion protein. Moreover, GrB has recently been found to be very stable in general, and it performs very well in the cleavage of different fusion proteins (Fynbo et al., Protein Expr. Purif. 39:209 (2005)).

Any member of the granzyme family of serine proteases, e.g., granzyme A and granzyme M, may be used as the recombinant protease component of the protease fusion in this invention. For example, granzyme M (GrM) is specifically found in the granules of natural killer cells and can hydrolyze the peptide sequence KV(Y)PL(M) with high efficiency and specificity (Mahrus et al., J: Biol. Chem. 279:54275 (2004)).

In designing and utilizing protease fusions of the invention, it should be noted that proteinase inhibitors may hamper the proteolytic activities of protease fusion proteins. For example, GrB is specifically inhibited by intracellular proteinase inhibitor 9 (PI-9), a member of the serpin superfamily that primarily exists in cytotoxic lymphocytes (Sun et al., J. Biol. Chem. 271:27802 (1996)) and has been detected in human plasma. GrB can also be inhibited by α₁-protease inhibitor (α₁PI) that is present in human plasma (Poe et al., J. Biol. Chem. 266:98 (1991)). GrM is inhibited by α₁-antichymotrypsin (ACT) and α₁PI (Mahrus et al., J. Biol. Chem. 279:54275 (2004)), and GrA is inhibited in vitro by protease inhibitors antithrombin III (ATIII) and α2-macroglobulin (α₂M) (Spaeny-Dekking et al., Blood 95:1465 (2000)). These proteinase inhibitors are also present in human plasma (Travis and Salvesen, Annu. Rev. Biochem. 52:655 (1983)).

One approach to preserve proteolytic activities of granzymes is to utilize complexation with proteoglycan, since the mature and active form of GrA has been observed in human plasma as a complex with serglycin, a granule-associated proteoglycan (Spaeny-Dekking et al., Blood 95:1465 (2000)). Glycosaminglycan complexes of GrB have also been found proteolytically active (Galvin et al., J. Immunol. 162:5345 (1999)). Thus, it may be possible to keep granzyme fusion proteins active in plasma through formulations using chondroitin sulfates.

Cathepsins and Caspases

Any member of the cathepsins (Chwieralski et al., Apoptosis 11:143 (2006)), e.g., cathepsin A, B, C, D, E, F, G, H, K, L, S, W, and X, may also be used as the recombinant protease for the present invention. Cathepsins are proteases that are localized intralysosomally under physiologic conditions, and therefore have optimum activity in acidic environments. Cathepsins comprise proteases of different enzyme classes; e.g., cathepsins A and G are serine proteases, cathepsins D and E are aspartic proteases. Certain cathepsins are caspases, a unique family of cysteine proteases that play a central role in the initiation and execution phases of apoptosis. Among all known mammalian proteases, only the serine protease granzyme B has substrate specificity similar to the caspases.

A cathepsin or caspase can be used as an exogenous activator or proactivator only if the protoxin to be activated is not exposed to that cathepsin or caspase prior to internalization (in the case of toxins that must be internalized) or during the course of the natural formation of the active toxin. For example, the protoxins of pore-forming toxins are activated at the cell surface, followed by oligomerization and pore formation. Because pore forming toxins do not localize to lysosome, cathepsins and caspases can be applied as exogenous activators. On the other hand, because the A-B toxin DT is known to be translocated directly into the cytosol through the endosome and/or lysosome, where cathepsins naturally reside, cathepsins should not be used as exogenous activators for DT-based protoxins. Other A-B toxins such as PEA may be compatible with the use of lysosomal proteases as exogenous activators, because they are transported to the trans-Golgi network and the ER before the translocation into cytosol. The bacterial toxins that can utilize cathepsins or other lysosomal proteases as exogenous activators include, but not limited to, PEA, shiga toxin, cholera toxin, and pertussis toxin. The bacterial toxins that are not suited for such use include DT, anthrax toxin, and clostridial neurotoxins (Falnes and Sandvig, Curr. Opin. Cell Biol. 2000, 12(4):407-13).

All caspases, including caspase-1, -2, -3, -4, -5, -6, -7, -8, -9 and more, show high selectivity and cleave proteins adjacent to an aspartate residue (Timmer and Salvesen, Cell Death Diff. 14:66-72 (2007)). The preferred cleavage site for caspase-1, 4, -5, and -14 are (W/Y)EXD↓Φ, where X is any residue and Φ represents a Gly, Ala, Thr, Ser, or Asn (SEQ ID NO:50). The preferred substrate for caspase-8, -9, and -10 contains the sequence of (I/L)EXD↓Φ (SEQ ID NO:51), and that of caspase-3 and -7 contains DEXD↓Φ (SEQ ID NO:52). Caspase-6 preferably cleaves at VEXD↓Φ (SEQ ID NO:53), while caspase-2 selectively targets (V/L)DEXD↓Φ(SEQ ID NO:54). Because the naturally occurring inhibitors of caspases, e.g., IAPs, are usually located intracellularly (LeBlanc, Prog. Neuropsychopharmacol. Biol. Psychiatry 27:215 (2003)), the probability of inhibition in plasma is dramatically reduced. Although caspase-1 and caspase-4 can be inhibited by PI-9 at moderate rates, it does not inhibit caspase-3 (Annand et al., Biochem. J. 342:655 (1999)).

Other Human Proteases

Many human proteases, including those have been identified as certain disease markers secreted by diseased cells, or associated with cancer invasion and metastasis, may be useful for the present invention as the heterologous protease. These proteases are well studied and detailed information on proteolytic activity and sequence selectivity is available. Examples of such proteases include urokinase plasminogen activator (uPA), which recognizes and cleaves GSGR↓SA (SEQ ID NO:55); prostate-specific antigen (PSA), which prefers substrate sequence SS(Y/F)Y↓SG (SEQ ID NO:56); renin, which cleaves at HPFHL↓VIH (SEQ ID NO:57); and MMP-2, which can cleave at HPVG↓LLAR (SEQ ID NO:58). Additional examples include the caspases, elastase, kallikreins, the matrix metalloprotease (MMP) family, the plasminogen activator family, as well as fibroblast activation protein.

In certain cases, the protease involved in one disease may be useful for the treatment of another disease that does not usually involve its overexpression. In other instances, the concentration of the secreted protease at native level may not be sufficient to activate corresponding toxin fusion to the extent that is necessary for targeted cell killing, i.e., is not operably present on the targeted cells. Additional proteolytic activity delivered to the cells through targeted protease fusion would provide desired toxin activation. In one embodiment, the protease fusion could have the same sequence specificity as the protease secreted by the diseased cells. In another embodiment, it may be desirable to use a combination of multiple, different, proteolytic cleavage activities to increase overall cleavage efficiency, with at least one of the proteolytic activity being provided by a targeted protease fusion.

Additional examples of endogenous proteases include those have been identified as certain disease markers, which are upregulated in certain disease. Non-limiting examples of such proteases include urokinase plasminogen activator (uPA), which recognizes and cleaves GSGR↓SA (SEQ ID NO:55); prostate-specific antigen (PSA), which prefers substrate sequence SS(Y/F)Y↓SG (SEQ ID NO:56); renin, which cleaves at HPFHL↓VIH (SEQ ID NO:57); and MMP-2, which can cleave at HPVG↓LLAR (SEQ ID NO:58).

Alternatively, potential candidate proteases may be screened in vitro by interactions with known proteinase inhibitors in plasma or with human plasma directly to avoid potential complications posed by these proteinase inhibitors. Alternatively, proteases for which cognate inhibitors are found in plasma can be engineered to provide mutant forms that resist inhibition. For example, in vitro E. coli expression-screening methods have been developed to select mutant proteases that are resistant to known HIV-1 protease inhibitors (Melnick et al., Antimicrob. Agents Chemother. 42:3256 (1998)).

Retroviral proteases may also be used for the present invention. Human retroviral proteases, including that of human immunodeficiency virus type 1 (HIV-1) (Beck et al., 2002), human T cell leukemia viruses (HTLV) (Shuker et al., Chem. Biol. 10:373 (2003)), and have been extensively studied as targets of anti-viral therapy. These proteases often have long recognition sequences and high substrate selectivity.

Picornaviral proteases may also be used for the present invention. Such picornaviral proteases have been studied as targets of anti-viral therapy, for example human Rhinovirus (HRV) (Binford et al., Antimicrob. Agents Chemother. 49:619 (2005)).

Recombinant heterologous proteases of any origin may be engineered to possess the aforementioned qualities and be used for the present invention. For example, tobacco etch virus (TEV) protease has very high substrate specificity and catalytic efficiency, and is used widely as a tool to remove peptide tags from recombinant proteins (Nunn et al., J. Mol. Biol. 350:145 (2005)). TEV protease recognizes an extended seven amino acid residue long consensus sequence E-X-X-Y-X-Q↓S/G (where X is any residue) (SEQ ID NO:59) that is present at protein junctions. Those skilled in the art would recognize that it is possible to engineer a particular protease such that its sequence specificity is altered to prefer another substrate sequence (Tozser et al., FEBS J. 272:514 (2005)).

Further modifications can be engineered to increase the activity and/or specificity of proteases. These modifications include PEGylation to increase stability to serum or to lower immunogenicity, and genetic engineering/selection may produce mutant proteases that possess altered properties such as resistance to certain inhibitors, increased thermal stability, and improved solubility.

Additional human proteases are set forth in Table 2.

MEROPS Clan Family ID Peptidase or homologue (subtype) MERNUM Gene Link Locus AA A1 A01.001 pepsin A MER00885 PGA3 5220 11q13 A01.003 gastricsin MER00894 PGC 5225 6p21.3-p21.1 A01.004 memapsin-2 MER05870 BACE1 23621 11q23.3-q24.1 A01.006 chymosin MER02929 CYMP 1542 1 A01.007 renin MER00917 REN 5972 1q32 A01.009 cathepsin D MER00911 CTSD 1509 11p15.5 A01.010 cathepsin E MER00944 CTSE 1510 1q31 A01.041 memapsin-1 MER05534 BACE2 25825 21pter-qter A01.046 napsin A MER04981 NAPSA 9476 19q13.33 A01.057 Mername-AA034 peptidase (deduced from nucleotide MER14038 1q23.3-24.3 sequence by MEROPS) A01.071 pepsin A5 (Homo sapiens) MER37291 PGA5 5222 11q13 A01.P01 napsin B pseudogene (napsin B pseudogene) MER04982 NAPSB 256236 19q13.33 A2 A02.010 mouse mammary tumor virus retropepsin (deduced from MER48030 nucleotide sequence by MEROPS) A02.011 human endogenous retrovirus K retropepsin (deduced from MER47534 5 nucleotide sequence by MEROPS) human endogenous retrovirus K retropepsin MER49453 human endogenous retrovirus K retropepsin MER00968 7 A02.019 multiple-sclerosis-associated retrovirus retropepsin MER47079 16 (deduced from nucleotide sequence by MEROPS) multiple-sclerosis-associated retrovirus retropepsin MER47096 4 (deduced from nucleotide sequence by MEROPS) multiple-sclerosis-associated retrovirus retropepsin MER47119 19 (deduced from nucleotide sequence by MEROPS) multiple-sclerosis-associated retrovirus retropepsin MER47124 7 (deduced from nucleotide sequence by MEROPS) multiple-sclerosis-associated retrovirus retropepsin MER47138 7 (deduced from nucleotide sequence by MEROPS) multiple-sclerosis-associated retrovirus retropepsin MER47145 2 (deduced from nucleotide sequence by MEROPS) multiple-sclerosis-associated retrovirus retropepsin MER47153 19 (deduced from nucleotide sequence by MEROPS) multiple-sclerosis-associated retrovirus retropepsin MER47162 5 (deduced from nucleotide sequence by MEROPS) multiple-sclerosis-associated retrovirus retropepsin MER47241 4 (deduced from nucleotide sequence by MEROPS) multiple-sclerosis-associated retrovirus retropepsin MER47244 15q21 (deduced from nucleotide sequence by MEROPS) multiple-sclerosis-associated retrovirus retropepsin MER47256 8 (deduced from nucleotide sequence by MEROPS) multiple-sclerosis-associated retrovirus retropepsin MER47257 8 (deduced from nucleotide sequence by MEROPS) multiple-sclerosis-associated retrovirus retropepsin MER47264 11 (deduced from nucleotide sequence by MEROPS) multiple-sclerosis-associated retrovirus retropepsin MER47271 12 (deduced from nucleotide sequence by MEROPS) multiple-sclerosis-associated retrovirus retropepsin MER47313 3 (deduced from nucleotide sequence by MEROPS) multiple-sclerosis-associated retrovirus retropepsin MER47390 2 (deduced from nucleotide sequence by MEROPS) multiple-sclerosis-associated retrovirus retropepsin MER47402 3 (deduced from nucleotide sequence by MEROPS) multiple-sclerosis-associated retrovirus retropepsin MER47412 3 (deduced from nucleotide sequence by MEROPS) multiple-sclerosis-associated retrovirus retropepsin MER47446 8 (deduced from nucleotide sequence by MEROPS) multiple-sclerosis-associated retrovirus retropepsin MER29837 (deduced from nucleotide sequence by MEROPS) multiple-sclerosis-associated retrovirus retropepsin MER47480 3 (deduced from nucleotide sequence by MEROPS) multiple-sclerosis-associated retrovirus retropepsin MER47492 2 (deduced from nucleotide sequence by MEROPS) multiple-sclerosis-associated retrovirus retropepsin MER47510 5 (deduced from nucleotide sequence by MEROPS) multiple-sclerosis-associated retrovirus retropepsin MER48013 (deduced from nucleotide sequence by MEROPS) A02.024 rabbit endogenous retrovirus endopeptidase MER43650 A02.053 S71-related human endogenous retropepsin MER01812 A02.055 RTVL-H-like putative peptidase (deduced from nucleotide MER47133 sequence by MEROPS) RTVL-H-like putative peptidase (deduced from nucleotide MER47160 19 sequence by MEROPS) RTVL-H-like putative peptidase (deduced from nucleotide MER47253 19 sequence by MEROPS) RTVL-H-like putative peptidase (deduced from nucleotide MER47260 3 sequence by MEROPS) RTVL-H-like putative peptidase (deduced from nucleotide MER47418 4 sequence by MEROPS) RTVL-H-like putative peptidase (deduced from nucleotide MER47440 1p33-p32 sequence by MEROPS) RTVL-H-like putative peptidase (pseudogene) MER15446 387590 22q11.2 A02.056 human endogenous retrovirus retropepsin homologue 1 MER15479 (deduced from ESTs by MEROPS) A02.057 human endogenous retrovirus retropepsin homologue 2 MER15481 (deduced from ESTs by MEROPS) A02.P01 endogenous retrovirus retropepsin pseudogene 1 (Homo MER29977 14q32.33 sapiens chromosome 14) (deduced from nucleotide sequence by MEROPS) A02.P02 endogenous retrovirus retropepsin pseudogene 2 MER29665 8p21.3-p22 (Homo sapiens chromosome 8) (deduced from nucleotide sequence by MEROPS) A02.P03 endogenous retrovirus retropepsin pseudogene 3 MER02660 17 (Homo sapiens chromosome 17) endogenous retrovirus retropepsin pseudogene 3 MER30286 (Homo sapiens chromosome 17) (deduced from nucleotide sequence by MEROPS) endogenous retrovirus retropepsin pseudogene 3 MER47144 2 (Homo sapiens chromosome 17) (deduced from nucleotide sequence by MEROPS) A02.P04 endogenous retrovirus retropepsin pseudogene 5 MER29664 12q13.1 (Homo sapiens chromosome 12) (deduced from nucleotide sequence by MEROPS) A02.P05 endogenous retrovirus retropepsin pseudogene 6 MER02094 7 (Homo sapiens chromosome 7) (deduced from nucleotide sequence by MEROPS) A02.P06 endogenous retrovirus retropepsin pseudogene 7 MER29776 6p21.3 (Homo sapiens chromosome 6) (deduced from nucleotide sequence by MEROPS) A02.P07 endogenous retrovirus retropepsin pseudogene 8 MER30291 Y (Homo sapiens chromosome Y) (deduced from nucleotide sequence by MEROPS) A02.P08 endogenous retrovirus retropepsin pseudogene 9 MER29680 19 (Homo sapiens chromosome 19) (deduced from nucleotide sequence by MEROPS) A02.P09 endogenous retrovirus retropepsin pseudogene 10 (Homo MER02848 12q23.3 sapiens chromosome 12) (deduced from nucleotide sequence by MEROPS) A02.P10 endogenous retrovirus retropepsin pseudogene 11 (Homo MER04378 17 sapiens chromosome 17) (deduced from nucleotide sequence by MEROPS) A02.P11 endogenous retrovirus retropepsin pseudogene 12 (Homo MER03344 11q11 sapiens chromosome 11) (deduced from nucleotide sequence by MEROPS) A02.P12 endogenous retrovirus retropepsin pseudogene 13 (Homo MER29779 2 sapiens chromosome 2 and similar) (deduced from nucleotide sequence by MEROPS) A02.P13 endogenous retrovirus retropepsin pseudogene 14 (Homo MER29778 2 sapiens chromosome 2) (deduced from nucleotide sequence by MEROPS) A02.P14 endogenous retrovirus retropepsin pseudogene 15 (Homo MER47158 19 sapiens chromosome 4) (deduced from nucleotide sequence by MEROPS) endogenous retrovirus retropepsin pseudogene 15 (Homo MER47332 3 sapiens chromosome 4) (deduced from nucleotide sequence by MEROPS) endogenous retrovirus retropepsin pseudogene 15 (Homo MER03182 4 sapiens chromosome 4) (deduced from nucleotide sequence by MEROPS) A02.P15 endogenous retrovirus retropepsin pseudogene 16 (deduced MER47165 19 from nucleotide sequence by MEROPS) endogenous retrovirus retropepsin pseudogene 16 (deduced MER47178 Y from nucleotide sequence by MEROPS) endogenous retrovirus retropepsin pseudogene 16 (deduced MER47200 19 from nucleotide sequence by MEROPS) endogenous retrovirus retropepsin pseudogene 16 (deduced MER47315 10 from nucleotide sequence by MEROPS) endogenous retrovirus retropepsin pseudogene 16 (deduced MER47405 8 from nucleotide sequence by MEROPS) endogenous retrovirus retropepsin pseudogene 16 (deduced MER30292 4 from nucleotide sequence by MEROPS) A02.P16 endogenous retrovirus retropepsin pseudogene 17 (Homo MER05305 8 sapiens chromosome 8) (deduced from nucleotide sequence by MEROPS) A02.P17 endogenous retrovirus retropepsin pseudogene 18 (Homo MER30288 4 sapiens chromosome 4) (deduced from nucleotide sequence by MEROPS) A02.P18 endogenous retrovirus retropepsin pseudogene 19 (Homo MER01740 16p11.2 sapiens chromosome 16) (deduced from nucleotide sequence by MEROPS) A02.P19 endogenous retrovirus retropepsin pseudogene 21 (Homo MER47222 11 sapiens) (deduced from nucleotide sequence by MEROPS) endogenous retrovirus retropepsin pseudogene 21 (Homo MER47454 3p24.3 sapiens) (deduced from nucleotide sequence by MEROPS) endogenous retrovirus retropepsin pseudogene 21 (Homo MER47477 4 sapiens) (deduced from nucleotide sequence by MEROPS) endogenous retrovirus retropepsin pseudogene 21 (Homo MER04403 sapiens) (deduced from nucleotide sequence by MEROPS) A02.P20 endogenous retrovirus retropepsin pseudogene 22 (Homo MER30287 Xq22.1 sapiens chromosome X) (deduced from nucleotide sequence by MEROPS) non- subfamily A2A non-peptidase homologues (deduced from MER47046 9q32 peptidase nucleotide sequence by MEROPS) homologue subfamily A2A non-peptidase homologues MER47052 6q21 subfamily A2A non-peptidase homologues (deduced from MER47076 X nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47080 19 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47088 Xq23 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47089 14q24.3 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47091 11 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47092 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47093 7 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47094 2 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47097 2 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47099 7q31.3 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47101 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47102 17 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47107 7 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47108 4p16 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47109 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47110 X nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47111 17 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47114 18 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47118 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47121 X nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47122 4p16 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47126 Y nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47129 7 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47130 Y nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47134 12p13 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47135 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47137 12p13 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47140 16 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47141 3 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47142 Y nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47148 2 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47149 3q26.2-27 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47151 5 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47154 5 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47155 5 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47156 5 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47157 19 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47159 19 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47161 19 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47163 5 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47166 2 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47171 18 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47173 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47174 2 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47179 2 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47183 Y nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47186 19 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47190 19 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47191 19 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47196 Y nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47198 10q22.3 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47199 19 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47201 19 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47202 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47203 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47204 8 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47205 Y nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47207 3 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47208 12p11.22 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47210 2 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47211 3 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47212 5 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47213 5 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47215 15q25 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47216 10p11.2-q21 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47218 8 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47219 11p14.3 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47221 15q21.3 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47224 2 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47225 2q33 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47226 8 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47227 8 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47230 10 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47232 7 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47233 16 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47234 11p15.4 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47236 2 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47238 2 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47239 7 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47240 2 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47242 4 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47243 4 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47249 5 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47251 18 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47252 12p13 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47254 17 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47255 15q15 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47263 5 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47265 12 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47266 10 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47267 5 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47268 3 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47269 5 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47272 3 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47273 10 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47274 10q23.32 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47275 3 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47276 3 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47279 5 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47280 5 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47281 5 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47282 5 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47284 15q26.2 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47285 11q11 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47289 16 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47290 2 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47294 2 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47295 3p nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47298 2 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47300 2 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47302 8 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47304 15q15 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47305 11p15 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47306 3 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47307 3 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47310 Y nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47311 3 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47314 2 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47318 2 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47320 Xp nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47321 2 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47322 7 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47326 12 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47327 Xp nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47330 4 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47333 18 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47362 15 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47366 8 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47369 11 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47370 18 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47371 18 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47375 11p15.2-p15.1 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47376 15q22-q24 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47381 Xq23 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47383 15 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47384 7 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47385 12p13 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47388 3 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47389 3 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47391 12p nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47394 2 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47396 2 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47400 12 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47401 3 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47403 3 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47406 2 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47407 1 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47410 5 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47411 5 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47413 1q42.12 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47414 8 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47416 4 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47417 4 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47420 5 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47423 4 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47424 4 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47428 4 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47429 4 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47431 4 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47434 2 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47439 7 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47442 11 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47445 18 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47449 8 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47450 8 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47452 1q44 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47455 4 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47457 4 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47458 3 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47459 8 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47463 4 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47468 4 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47469 4 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47470 3 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47476 4 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47478 5 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47483 16 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47488 2 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47489 4 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47490 2 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47493 3 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47494 5 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47495 4 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47496 4 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47497 4 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47499 11p15.4 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47502 3 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47504 3 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47511 5 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47513 5 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47514 5 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47515 11p11.2 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47516 4 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47520 X nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47533 3 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47537 3 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47569 3 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47570 3 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47584 3 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47603 4 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47604 5 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47606 12q15-q21 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47609 3 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47616 3 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47619 5 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47648 5 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47649 16 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER47662 12q24.11 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER48004 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER48018 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER48019 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER48023 21q21 nucleotide sequence by MEROPS) subfamily A2A non-peptidase homologues (deduced from MER48037 8q21-q23 nucleotide sequence by MEROPS) unassigned subfamily A2A unassigned peptidases (deduced from MER47117 7 nucleotide sequence by MEROPS) subfamily A2A unassigned peptidases (deduced from MER47164 19 nucleotide sequence by MEROPS) subfamily A2A unassigned peptidases (deduced from MER47206 Y nucleotide sequence by MEROPS) subfamily A2A unassigned peptidases (deduced from MER47231 16 nucleotide sequence by MEROPS) subfamily A2A unassigned peptidases (deduced from MER47291 8 nucleotide sequence by MEROPS) subfamily A2A unassigned peptidases (deduced from MER47386 5 nucleotide sequence by MEROPS) subfamily A2A unassigned peptidases (deduced from MER47479 X nucleotide sequence by MEROPS) subfamily A2A unassigned peptidases (deduced from MER47559 12 nucleotide sequence by MEROPS) subfamily A2A unassigned peptidases (deduced from MER47583 16 nucleotide sequence by MEROPS) AD A22 A22.001 presenilin 1 MER05221 PSEN1 5663 14q24.3 A22.002 presenilin 2 MER05223 PSEN2 5664 1q31-q42 A22.003 impas 1 peptidase MER19701 HM13 81502 20q11.21 A22.004 impas 4 peptidase MER19715 56928 19p13.3 A22.005 impas 2 peptidase MER19708 121665 12q24.31 A22.006 impas 5 peptidase MER19712 162540 17q21.31 A22.007 impas 3 peptidase MER19711 84888 15q21.2 A22.P01 possible family A22 pseudogene (Homo sapiens MER29974 18 chromosome 18) (deduced from nucleotide sequence by MEROPS) A22.P02 possible family A22 pseudogene (Homo sapiens MER23159 11q12.2 chromosome 11) CA C1 C01.009 cathepsin V MER04437 CTSL2 1515 9q22.2 C01.013 cathepsin X MER04508 CTSZ 1522 20q13 C01.014 cathepsin L-like peptidase 2 MER05210 CTSLL2 1517 10q C01.015 cathepsin L-like peptidase 3 MER05209 CTSLL3 1518 10q22.3-q23.1 C01.018 cathepsin F MER04980 CTSF 8722 11q13.1-q13.3 C01.032 cathepsin L MER00622 CTSL 1514 9q21-q22 C01.034 cathepsin S MER00633 CTSS 1520 1q21 C01.035 cathepsin O MER01690 CTSO 1519 4q31-q32 C01.036 cathepsin K MER00644 CTSK 1513 1q21 C01.037 cathepsin W MER03756 CTSW 1521 11q13.1 C01.040 cathepsin H MER00629 CTSH 1512 15q24-q25 C01.060 cathepsin B MER00686 CTSB 1508 8p22 C01.070 dipeptidyl-peptidase I MER01937 CTSC 1075 11q14.1-q14.3 C01.084 bleomycin hydrolase (animal) MER02481 BLMH 642 17q11.1-q11.2 C01.973 tubulointerstitial nephritis antigen MER16137 TINAG 27283 6p11.2p12 C01.975 tubulointerstitial nephritis antigen-related protein MER21799 LCN7 64129 1p34.3 C01.P02 cathepsin L-like pseudogene 1 (Homo sapiens) MER02789 CTSLL1 1516 10q (pseudogene) C01.P03 cathepsin B-like pseudogene (chromosome 4, MER29469 4 Homo sapiens) C01.P04 cathepsin B-like pseudogene (chromosome 1, MER29457 1q42.11 (Homo sapiens) C2 C02.001 calpain-1 MER00770 CAPN1 823 11q13 C02.002 calpain-2 MER00964 CAPN2 824 1q41-q42 C02.004 calpain-3 MER01446 CAPN3 825 15q15.1-q21.1 C02.006 calpain-9 MER04042 CAPN9 10753 1q42.11-q42.3 C02.007 calpain-8 MER21474 1q41 C02.008 calpain-7 MER05537 CAPN7 23473 3p24 C02.010 calpain-15 MER04745 SOLH 6650 16p13.3 C02.011 calpain-5 MER02939 CAPN5 726 11q14 C02.013 calpain-11 MER05844 CAPN11 11131 6p12 C02.017 calpain-12 (deduced from nucleotide sequence MER29889 CAPN12 147968 19q13.2 by MEROPS) C02.018 calpain-10 MER13510 CAPN10 11132 2q37.3 C02.020 calpain-13 MER20139 CAPN13 92291 2p21-22 C02.021 calpain-14 MER29744 CAPN14 114773 2p23.1-p21 C02.971 calpamodulin (calpamodulin) MER00718 CAPN6 827 Xq23 C02.972 hypothetical protein flj40251 MER03201 C6orf103 79747 6q24.2 C12 C12.001 ubiquitinyl hydrolase-L1 MER00832 UCHL1 7345 4p14 C12.003 ubiquitinyl hydrolase-L3 MER00836 UCHL3 7347 13q21.2-q22.1 C12.004 ubiquitinyl hydrolase-BAP1 (KIAA0272 protein) MER03989 BAP1 8314 3p21.2-p21.31 C12.005 ubiquitinyl hydrolase-UCH37 MER05539 UCHL5 51377 1q32 CD C13 C13.002 legumain (plant alpha form) MER44591 C13.004 legumain MER01800 LGMN 5641 14q32.1 C13.005 glycosylphosphatidylinositol:protein transamidase MER02479 PIGK 10026 1 C13.P01 legumain pseudogene (Homo sapiens) MER29741 LGMN2P 122199 13q21.2 C14 C14.001 caspase-1 MER00850 CASP1 834 11q22.2-q22.3 C14.003 caspase-3 MER00853 CASP3 836 4q33-q35.1 C14.004 caspase-7 MER02705 CASP7 840 10q25.1-q25.2 C14.005 caspase-6 MER02708 CASP6 839 4q25 C14.006 caspase-2 MER01644 CASP2 835 7q34-q35 C14.007 caspase-4 MER01938 CASP4 837 11q22.2-q22.3 C14.008 caspase-5 MER02240 CASP5 838 11q22.2-q22.3 C14.009 caspase-8 MER02849 CASP8 841 2q33-q34 C14.010 caspase-9 MER02707 CASP9 842 1p36.1-p36.3 C14.011 caspase-10 MER02579 CASP10 843 2q33-q34 C14.018 caspase-14 MER12083 CASP14 23581 19p13.1 C14.026 paracaspase MER19325 MALT1 10892 18q21 C14.028 Mername-AA143 peptidase MER21304 11q22.3 C14.029 Mername-AA186 peptidase MER20516 11q22.3 C14.032 putative caspase (Homo sapiens) MER21463 C14.971 FLIP protein (casper) MER03026 CFLAR 8837 2q33-q34 C14.976 Mername-AA142 protein MER21316 11q22.3 C14.P01 caspase-12 pseudogene (Homo sapiens) MER19698 CASP12P1 120329 11q22.3 C14.P02 Mername-AA093 caspase pseudogene MER14766 197350 16p13.3 CF C15 C15.010 pyroglutamyl-peptidase I (chordate) MER11032 PGPEP1 54858 19p13.11 C15.011 Mername-AA073 peptidase (deduced from MER29978 145814 15q26.3 nucleotide sequence by MEROPS) CA C19 C19.001 ubiquitin-specific peptidase 5 MER02066 USP5 8078 12p13 C19.009 ubiquitin-specific peptidase 6 MER00863 USP6 9098 17q11 C19.010 ubiquitin-specific peptidase 4 (ubiquitin carboxy-terminal MER01795 USP4 7375 3p21.31 hydrolase UNP) C19.011 ubiquitin-specific peptidase 8 (KIAA0055 protein) MER01884 USP8 9101 15q11.2-q21.1 C19.012 ubiquitin-specific peptidase 13 MER02627 USP13 8975 3q26.2-q26.3 C19.013 ubiquitin-specific peptidase 2 MER04834 USP2 9099 11q23.3 C19.014 ubiquitin-specific peptidase 11 MER02693 USP11 8237 Xp11.23 C19.015 ubiquitin-specific peptidase 14 MER02667 USP14 9097 18p11.32 C19.016 ubiquitin-specific peptidase 7 (ubiquitin carboxyl-terminal MER02896 USP7 7874 16p13.3 hydrolase HAUSP) C19.017 ubiquitin-specific peptidase 9X MER05877 USP9X 8239 Xp11.4 C19.018 ubiquitin-specific peptidase 10 (KIAA0190 protein) MER04439 USP10 9100 16q23.1 C19.019 ubiquitin-specific peptidase 1 MER04978 USP1 7398 1p31.3-p32.1 C19.020 ubiquitin-specific peptidase 12 MER05454 USP12 9959 5q33-q34 C19.021 ubiquitin-specific peptidase 16 MER05493 USP16 10600 21q22.11 C19.022 ubiquitin-specific peptidase 15 MER05427 USP15 9958 12q14 C19.023 ubiquitin-specific peptidase 17 MER02900 USP17 23661 4p15 C19.024 ubiquitin-specific peptidase 19 MER05428 USP19 10869 3p21.31 C19.025 ubiquitin-specific peptidase 20 MER05494 USP20 10868 9q34.13 C19.026 ubiquitin-specific peptidase 3 MER05513 USP3 9960 15q22.3 C19.028 ubiquitin-specific peptidase 9Y MER04314 USP9Y 8287 Yq11.2 C19.030 ubiquitin-specific peptidase 18 MER05641 USP18 11274 22q11.21 C19.034 ubiquitin-specific peptidase 21 MER06258 USP21 27005 1q22 C19.035 ubiquitin-specific peptidase 22 MER12130 USP22 23326 17p13.2 C19.037 ubiquitin-specific peptidase 33 MER14335 USP33 23032 1p31.1 C19.040 ubiquitin-specific peptidase 29 MER12093 USP29 57663 19q13.43 C19.041 ubiquitin-specific peptidase 25 MER11115 USP25 29761 21q11.2 C19.042 ubiquitin-specific peptidase 36 MER14033 USP36 57602 17q25.3 C19.044 ubiquitin-specific peptidase 32 MER14290 USP32 84669 17q23.3 C19.046 ubiquitin-specific peptidase 26 (human-type) MER14292 USP26 83844 Xq26.2 C19.047 ubiquitin-specific peptidase 24 MER05706 USP24 23358 1p32.1 C19.048 ubiquitin-specific peptidase 42 MER11852 USP42 84132 7p22.2 C19.052 ubiquitin-specific peptidase 46 MER14629 USP46 64854 4q11 C19.053 ubiquitin-specific peptidase 37 MER14633 USP37 57695 2q36.1 C19.054 ubiquitin-specific peptidase 28 MER14634 USP28 57646 11q23 C19.055 ubiquitin-specific peptidase 47 MER14636 USP47 55031 11p15.2 C19.056 ubiquitin-specific peptidase 38 MER14637 USP38 84640 4q31.1 C19.057 ubiquitin-specific peptidase 44 MER14638 USP44 84101 12q21.33 C19.058 ubiquitin-specific peptidase 50 MER30315 USP50 373509 15q21.1 C19.059 ubiquitin-specific peptidase 35 MER14646 USP35 57558 11q13.5 C19.060 ubiquitin-specific peptidase 30 MER14649 USP30 84749 12q23.3 C19.062 Mername-AA091 peptidase (deduced from nucleotide MER14743 Xq21.31 sequence by MEROPS) C19.064 ubiquitin-specific peptidase 45 MER30314 USP45 85015 6q16.3 C19.065 ubiquitin-specific peptidase 51 MER14769 USP51 158880 Xp11.21-22 C19.067 ubiquitin-specific peptidase 34 MER14780 USP34 23021 2p15 C19.068 ubiquitin-specific peptidase 48 MER64620 USP48 84196 1p36.12 C19.069 ubiquitin-specific peptidase 40 MER15483 USP40 55230 2q37.1 C19.070 ubiquitin-specific peptidase 41 MER45268 USP41 150200 22q11.22 C19.071 ubiquitin-specific peptidase 31 MER15493 USP31 57478 16p12.3 C19.072 Mername-AA129 peptidase (deduced from ESTs MER16485 by MEROPS) C19.073 ubiquitin-specific peptidase 49 MER16486 USP49 25862 6pter-p12.1 C19.075 Mername-AA187 peptidase MER52579 USP27X 373504 Xp11.23 C19.078 USP17-like peptidase MER30192 401447 8p23.1 C19.080 ubiquitin-specific peptidase 54 MER28714 USP54 159195 10q22.3 C19.081 ubiquitin-specific peptidase 53 MER27329 USP53 54532 4q27 C19.972 ubiquitin-specific endopeptidase 39 [misleading] MER64621 USP39 10713 2q11.2 C19.974 Mername-AA090 non-peptidase homologue (deduced from MER14739 22q11.2 nucleotide sequence by MEROPS) C19.976 ubiquitin-specific peptidase 43 [misleading] MER30140 USP43 124739 17p13.1 C19.978 ubiquitin-specific peptidase 52 [misleading] MER30317 USP52 9924 12q13.2-q13.3 C19.980 Mername-AA088 peptidase (deduced from nucleotide MER14750 USP8P 6p21.3 sequence by MEROPS) C19.P01 NEK2 pseudogene (deduced from nucleotide sequence by MER14736 NEK2P 326302 14q11.2 MEROPS) C19.P02 C19 pseudogene (Homo sapiens: chromosome 5) MER29972 5 (deduced from nucleotide sequence by MEROPS) PC C26 C26.001 gamma-glutamyl hydrolase MER02963 GGH 8836 8q12.23-q13.1 C26.950 guanine 5′-monophosphate synthetase MER43387 GMPS 8833 3q24 C26.951 carbamoyl-phosphate synthase (Homo sapiens) MER78640 (CPS1 protein) C26.952 dihydro-orotase (N-terminal unit) (Homo sapiens) MER60647 CAD 790 2p22-p21 PB C44 C44.001 amidophosphoribosyltransferase precursor MER03314 PPAT 5471 4q121 C44.970 glutamine-fructose-6-phosphate transaminase 1 MER03322 GFPT1 2673 2p13 (glucosamine-fructose-6-phosphate aminotransferase) C44.972 glutamine:fructose-6-phosphate amidotransferase MER12158 GFPT2 9945 5q34-q35 C44.973 Mername-AA144 protein MER21319 Xq13.3 C44.974 asparagine synthetase MER33254 ASNS 440 7q21.3 CH C46 C46.002 Sonic hedgehog protein MER02539 SHH 6469 7q36 C46.003 Indian hedgehog protein MER02538 IHH 3549 2 C46.004 Desert hedgehog protein MER12170 DHH 50846 12q12-13.1 CE C48 C48.002 SENP1 peptidase MER11012 SENP1 29843 12q13.1 C48.003 SENP3 peptidase MER11019 SENP3 26168 17p13 C48.004 SENP6 peptidase MER11109 SENP6 26054 6q13-q14.3 C48.007 SENP2 peptidase MER12183 SENP2 59343 3q28 C48.008 SENP5 peptidase MER14032 SENP5 205564 3q29 C48.009 SENP7 peptidase MER14095 SENP7 57337 3q12 C48.011 SENP8 peptidase MER16161 SENP8 123228 15q22.32 C48.012 SENP4 peptidase MER05557 CD C50 C50.001 separase MER11775 ESPL1 9700 8 C50.P01 separase-like pseudogene (deduced from nucleotide MER14797 8q21.2 sequence by MEROPS) CA C54 C54.002 autophagin-2 MER13564 ATG4A 115201 Xq22.1-22.3 C54.003 autophagin-1 MER13561 ATG4B 23192 2 C54.004 autophagin-3 MER14316 ATG4C 84938 1p31.3 C54.005 autophagin-4 MER64622 ATG4D 84971 19p13.2 PC C56 C56.002 DJ-1 putative peptidase MER03390 PARK7 11315 1p36.2-p36.3 C56.003 Mername-AA100 peptidase (deduced from MER14802 12q13 nucleotide sequence by MEROPS) C56.971 Mername-AA101 non-peptidase homologue (deduced from MER14803 9q22.32 nucleotide sequence by MEROPS) C56.972 KIAA0361 protein (Homo sapiens) MER42827 PFAS 5198 17p13.1 C56.974 FLJ34283 protein (Homo sapiens) MER44553 347862 11p15.5 CA C64 C64.001 Cezanne deubiquitinylating peptidase MER29042 ZA20D1 56957 1q21.3 C64.002 Cezanne-2 peptidase MER29044 C15orf16 161725 15q13.1 C64.003 tumor necrosis factor alpha-induced protein 3 MER29050 TNFAIP3 7128 6q23-q25 C64.004 TRABID protein MER29052 ZRANB1 54764 10q26.2 C65 C65.001 otubain-1 MER29056 OTUB1 55611 11q13.1 C65.002 otubain-2 MER29061 OTUB2 78990 14q32.13-q32.2 C67 C67.001 CylD protein MER30104 CYLD 1540 16q12.1 PB C69 C69.003 secernin 1 MER45376 SCRN1 9805 7p14.3-p14.1 C69.004 secernin 2 (SCRN2 protein) MER64573 SCRN2 90507 17q21.32 C69.005 secernin 3 (SCRN3 protein) MER64582 SCRN3 79634 2q31.1 CA C78 C78.001 UfSP1 peptidase MER42724 C78.002 UfSP2 peptidase MER60306 MA M1 M01.001 aminopeptidase N MER00997 ANPEP 290 15q25-q26 M01.003 aminopeptidase A MER01012 ENPEP 2028 4q25 M01.004 leukotriene A4 hydrolase (LTA4H protein) MER01013 LTA4H 4048 12q22 M01.008 pyroglutamyl-peptidase II MER12221 TRHDE 29953 12q15-q21 M01.010 cytosol alanyl aminopeptidase MER02746 NPEPPS 9520 17q12-q21 M01.011 cystinyl aminopeptidase MER02060 LNPEP 4012 5q15 M01.014 aminopeptidase B MER01494 RNPEP 6051 1q32.1-q32.2 M01.018 aminopeptidase PILS MER05331 51752 5q15 M01.022 Mername-AA050 peptidase MER12271 RNPEPL1 57140 2q37.3 M01.024 leukocyte-derived arginine aminopeptidase MER02968 64167 16 M01.026 laeverin MER52595 206338 5q23.1 M01.028 aminopeptidase O MER19730 C9orf3 84909 9q22.32 M01.972 Tata binding protein associated factor MER26493 TAF2 6873 8q24.12 M2 M02.001 angiotensin-converting enzyme peptidase unit 1 (peptidase MER04967 ACE 1636 17q23 unit 1) M02.004 angiotensin-converting enzyme peptidase unit 2 (peptidase MER01019 ACE 1636 17q23 unit 2) M02.006 angiotensin-converting enzyme 2 MER11061 ACE2 5972 Xp22 M02.972 Mername-AA153 protein MER20514 17q21.33 M3 M03.001 thimet oligopeptidase MER01737 THOP1 7064 19q13.3 M03.002 neurolysin MER10991 NLN 57486 5q12.3 M03.006 mitochondrial intermediate peptidase MER03665 MIPEP 4285 13q12 M03.971 Mername-AA154 protein MER21317 7q21.13 M8 M08.003 leishmanolysin-2 MER14492 LMLN 89782 3q29 M10 M10.001 matrix metallopeptidase-1 MER01063 MMP1 4312 11q22-q23 M10.002 matrix metallopeptidase-8 MER01084 MMP8 4317 11q21-q22 M10.003 matrix metallopeptidase-2 MER01080 MMP2 4313 16q13 M10.004 matrix metallopeptidase-9 MER01085 MMP9 4318 20q11.2-q13.1 M10.005 matrix metallopeptidase-3 MER01068 MMP3 4314 11q23 M10.006 matrix metallopeptidase-10 (human type) MER01072 MMP10 4319 11q22.3-q23 M10.007 matrix metallopeptidase-11 MER01075 MMP11 4320 22q11.2 M10.008 matrix metallopeptidase-7 MER01092 MMP7 4316 11q21-q22 M10.009 matrix metallopeptidase-12 MER01089 MMP12 4321 11q22.2-q22.3 M10.013 matrix metallopeptidase-13 MER01411 MMP13 4322 11q22.3 M10.014 membrane-type matrix metallopeptidase-1 MER01077 MMP14 4323 14q11-q12 M10.015 membrane-type matrix metallopeptidase-2 MER02383 MMP15 4324 16q13-q21 M10.016 membrane-type matrix metallopeptidase-3 MER02384 MMP16 4325 8q21 M10.017 membrane-type matrix metallopeptidase-4 MER02595 MMP17 4326 12q24.3 M10.019 matrix metallopeptidase-20 MER03021 MMP20 9313 11q22.3 M10.021 matrix metallopeptidase-19 MER02076 MMP19 4327 12q14 M10.022 matrix metallopeptidase-23B MER04766 MMP23B 8510 1p36.3 M10.023 membrane-type matrix metallopeptidase-5 MER05638 MMP24 10893 20q11.2 M10.024 membrane-type matrix metallopeptidase-6 MER12071 MMP25 64386 16p13.3 M10.026 matrix metallopeptidase-21 MER06101 MMP21 118856 10q26.2 M10.027 matrix metallopeptidase-22 MER14098 MMP27 64066 11q24 M10.029 matrix metallopeptidase-26 MER12072 MMP26 56547 11p15 M10.030 matrix metallopeptidase-28 MER13587 MMP28 79148 17q21.1 M10.037 matrix metallopeptidase-23A MER37217 MMP23A 8511 1p36.3 M10.950 macrophage elastase homologue (chromosome 8, Homo MER30035 8 sapiens) (deduced from nucleotide sequence by MEROPS) M10.971 Mername-AA156 protein MER21309 11q22.2 M10.973 matrix metallopeptidase-like 1 MER45280 MMPL1 4328 16p13.3 M12 M12.002 meprin alpha subunit (alpha) MER01111 MEP1A 4224 6p21.2-p21.1 M12.004 meprin beta subunit (beta) MER05213 MEP1B 4225 18q12.2-q12.3 M12.005 procollagen C-peptidase MER01113 BMP1 649 8p21 M12.016 mammalian tolloid-like 1 protein MER05124 TLL1 7092 4q32-q33 M12.018 mammalian tolloid-like 2 protein MER05866 TLL2 7093 10q23-q24 M12.021 ADAMTS9 peptidase MER12092 ADAMTS9 56999 3p14.2-p14.3 M12.024 ADAMTS14 peptidase MER16700 ADAMTS14 140766 10q2 M12.025 ADAMTS15 peptidase MER17029 ADAMTS15 170689 11q25 M12.026 ADAMTS16 peptidase MER15689 ADAMTS16 170690 5p15 M12.027 ADAMTS17 peptidase MER16302 ADAMTS17 170691 15q24 M12.028 ADAMTS18 peptidase MER16090 ADAMTS18 170692 16q23 M12.029 ADAMTS19 peptidase MER15663 ADAMTS19 171019 5q31 M12.201 ADAM1 peptidase MER03912 ADAM1 8759 12q24 M12.208 ADAM8 peptidase MER03902 ADAM8 101 10q26.3 M12.209 ADAM9 peptidase MER01140 ADAM9 8754 8p11.22 M12.210 ADAM10 peptidase MER02382 ADAM10 102 15q21.3 M12.212 ADAM12 peptidase MER05107 ADAM12 8038 10q26 M12.214 adamalysin-19 MER12241 ADAM19 8728 5q32-33 M12.215 ADAM15 peptidase MER02386 ADAM15 8751 1q21.3 M12.217 ADAM17 peptidase MER03094 ADAM17 6868 2p25 M12.218 ADAM20 peptidase MER04725 ADAM20 8748 14q24.1 M12.219 ADAMDEC1 peptidase MER00743 ADAMDEC1 27299 8p21.1 M12.220 ADAMTS3 peptidase MER05100 ADAMTS3 9508 4q21 M12.221 ADAMTS4 peptidase MER05101 ADAMTS4 9507 1q31-q32 M12.222 ADAMTS1 peptidase MER05546 ADAMTS1 9510 21q22-q22 M12.224 ADAM28 peptidase (human-type) MER05495 ADAM28 10863 8p21.2 M12.225 ADAMTS5 peptidase MER05548 ADAMTS5 11096 21q22.1-q22 M12.226 ADAMTS8 peptidase MER05545 ADAMTS8 11095 11q25 M12.230 ADAMTS6 peptidase MER05893 ADAMTS6 11174 5pter-qter M12.231 ADAMTS7 peptidase MER05894 ADAMTS7 11173 15pter-qter M12.232 ADAM30 peptidase MER06268 ADAM30 11085 1p11-p13 M12.234 ADAM21 peptidase (Homo sapiens) (ADAM 21 protein) MER04726 ADAM21 8747 14q24.1 M12.235 ADAMTS10 peptidase MER14331 ADAMTS10 81794 19p13.3 M12.237 ADAMTS12 peptidase MER14337 ADAMTS12 81792 5q35 M12.241 ADAMTS13 peptidase MER15450 ADAMTS13 11093 9q34 M12.244 ADAM33 peptidase MER15143 ADAM33 80332 20p13 M12.245 ovastacin MER29996 ASTL 431705 2q11.1 M12.246 ADAMTS20 peptidase (Homo sapiens) MER26906 ADAMTS20 80070 12q12 M12.301 procollagen I N-peptidase MER04985 ADAMTS2 9509 5q23-q24 M12.950 ADAM2 protein (ADAM 2 protein) MER03090 ADAM2 2515 8p11.2 M12.954 ADAM6 protein (deduced from nucleotide sequence by MER47044 14q32.33 MEROPS) ADAM6 protein (deduced from nucleotide sequence by MER47250 MEROPS) ADAM6 protein (deduced from nucleotide sequence by MER47654 16 MEROPS) M12.956 ADAM7 protein (GP-83 glycoprotein) MER05109 ADAM7 8756 8p21.2 M12.957 ADAM18 protein MER12230 ADAM18 8749 8p22 M12.960 ADAM32 protein MER26938 ADAM32 203102 8p11.21 M12.962 non-peptidase homologue (Homo sapiens chromosome 4) MER29973 (deduced from nucleotide sequence by MEROPS) M12.974 ADAM3A protein (human-type) (ADAM 3A protein) MER05200 ADAM3A 1587 8p21-p12 M12.975 ADAM3B protein (human-type) (ADAM 3B protein) MER05199 ADAM3B 1596 16q12.1 M12.976 ADAM11 protein (ADAM 11 protein) MER01146 ADAM11 4185 17q21.3 M12.978 ADAM22 protein (ADAM 22 protein) MER05102 ADAM22 53616 7q21 M12.979 ADAM23 protein (ADAM 23 protein) MER05103 ADAM23 8745 2q33 M12.981 ADAM29 protein MER06267 ADAM29 11086 4q34.2-qter M12.987 protein similar to ADAM21 peptidase preproprotein (Homo MER26944 sapiens) M12.990 Mername AA-225 peptidase homologue (Homo sapiens) MER47474 15 (deduced from nucleotide sequence by MEROPS) M12.P01 putative ADAM pseudogene (chromosome 4, MER29975 Homo sapiens) M13 M13.001 neprilysin MER01050 MME 4311 3q21-q27 M13.002 endothelin-converting enzyme 1 MER01057 ECE1 1889 1p36.1 M13.003 endothelin-converting enzyme 2 MER04776 ECE2 9718 3q26.1-q26.33 M13.007 DINE peptidase MER05197 ECEL1 9427 2q37.1 M13.008 neprilysin-2 MER13406 MELL1 79258 1p36 M13.090 Kell blood-group protein MER01054 KEL 3792 7q33 M13.091 PHEX peptidase MER02062 PHEX 5251 Xp22.2-p22.1 MC M14 M14.001 carboxypeptidase A1 MER01190 CPA1 1357 7q32 M14.002 carboxypeptidase A2 MER01608 CPA2 1358 7q32 M14.003 carboxypeptidase B MER01194 CPB1 1360 3q24 M14.004 carboxypeptidase N MER01198 CPN1 1369 10 M14.005 carboxypeptidase E MER01199 CPE 1363 4 M14.006 carboxypeptidase M MER01205 CPM 1368 12q15 M14.009 carboxypeptidase U MER01193 CPB2 1361 13q14.11 M14.010 carboxypeptidase A3 MER01187 CPA3 1359 3q21-q25 M14.011 metallocarboxypeptidase D peptidase unit 1 MER03781 CPD 1362 17p11.1-q11.2 (peptidase unit 1) M14.012 metallocarboxypeptidase Z MER03428 CPZ 8532 4p16.1 M14.016 metallocarboxypeptidase D peptidase unit 2 MER04963 CPD 1362 17p11.1-q11.2 (peptidase unit 2) M14.017 carboxypeptidase A4 MER13421 CPA4 51200 7q32 M14.018 carboxypeptidase A6 MER13456 CPA6 57094 8q12.3 M14.020 carboxypeptidase A5 MER17121 CPA5 93979 7q32 M14.021 metallocarboxypeptidase O MER16044 CPO 130749 2q34 M14.025 Mername-AA216 hypothetical peptidase MER33174 60509 2p23.3 M14.026 Mername-AA213 putative peptidase MER33176 AGBL3 340351 7q33 M14.027 hypothetical protein flj14442 (Homo sapiens) and similar MER33178 AGBL4 84871 1p33 M14.028 Mername-AA217 hypothetical peptidase MER33179 AGTPBP1 23287 9q22.1 M14.029 A430081C19RIK (Mus musculus)-type protein MER37713 AGBL2 79841 11p11.2 M14.950 metallocarboxypeptidase D non-peptidase unit MER04964 CPD 1362 17p11.1-q11.2 (peptidase unit 3) M14.951 adipocyte-enhancer binding protein 1 MER03889 AEBP1 165 7 M14.952 carboxypeptidase-like protein X1 MER13404 CPXM 56265 20p12.3-p13 M14.954 cytosolic carboxypeptidase MER26952 CPXM2 119587 10q26.13 ME M16 M16.002 insulysin MER01214 IDE 3416 10q23-q25 M16.003 mitochondrial processing peptidase MER04497 PMPCB 9512 7q22.1/ beta-subunit (beta) 7q22-q31.1 M16.005 nardilysin MER03883 NRD1 4898 1p32.2/ 1p32.2-p32.1 M16.009 eupitrilysin (MP1 protein) MER04877 PITRM1 10531 10p15.2 M16.971 mitochondrial processing peptidase non-peptidase alpha MER01413 PMPCA 23203 9q34.3 subunit (alpha) M16.973 ubiquinol-cytochrome c reductase core protein I (ubiquinol- MER03543 UQCRC1 7384 3p21.3 cytochrome c reductase core protein 1) M16.974 ubiquinol-cytochrome c reductase core protein II MER03544 UQCRC2 7385 16p12 (ubiquinol-cytochrome c reductase core protein 2) M16.976 Mername-AA158 protein MER21876 4q22.2 M16.980 mitochondrial processing peptidase beta subunit domain 2 MER43988 PMPCB 9512 7q22.1/ (beta) 7q22-q31.1 M16.981 ubiquinol-cytochrome c reductase core protein domain 2 MER43998 UQCRC1 7384 3p21.3 (ubiquinol-cytochrome c reductase core protein 1) M16.982 insulysin unit 2 MER46821 IDE 3416 10q23-q25 M16.983 nardilysin unit 2 MER46874 NRD1 4898 1p32.2/ 1p32.2-p32.1 M16.984 insulysin unit 3 (Homo sapiens) (IDE protein) MER78753 IDE 3416 10q23-q25 MF M17 M17.001 leucyl aminopeptidase (animal) MER03100 LAP3 51056 4p15.33 M17.005 Mername-AA040 peptidase MER03919 6 M17.006 Mername-AA014 peptidase MER13416 NPEPL1 79716 20q13.32 MH M18 M18.002 aspartyl aminopeptidase MER03373 DNPEP 23549 2q36.1 MJ M19 M19.001 membrane dipeptidase MER01260 DPEP1 1800 16q24.3 M19.002 membrane-bound dipeptidase-2 MER13499 DPEP2 64174 16q22.1 M19.004 membrane-bound dipeptidase-3 MER13496 DPEP3 64180 16q22.1 MH M20 M20.005 carnosine dipeptidase II MER14551 CNDP2 55748 18 M20.006 carnosine dipeptidase I (sequenced from cDNA by MER15142 CNDP1 84735 18q22.3 MEROPS) M20.011 Mername-AA218 hypothetical peptidase MER33182 148811 1q32.1 M20.971 Mername-AA161 protein MER21873 ACY1L2 135293 6q15 M20.973 aminoacylase (aminoacylase-1) MER01271 ACY1 95 3p21.1 MK M22 M22.003 Kael putative peptidase MER01577 OSGEP 55644 14q11.1 M22.004 Mername-AA018 peptidase MER13498 OSGEPL1 64172 2q32.3 MG M24 M24.001 methionyl aminopeptidase 1 MER01342 METAP1 23173 4q23 M24.002 methionyl aminopeptidase 2 MER01728 METAP2 10988 12q22 M24.005 aminopeptidase P2 MER04498 XPNPEP2 7512 Xq25 M24.007 Xaa-Pro dipeptidase (eukaryote) MER01248 PEPD 5184 19cen-q13.11 M24.009 aminopeptidase P1 MER04321 XPNPEP1 7511 10q25.3 M24.026 aminopeptidase P homologue MER13463 63929 22q13.31-q13.33 M24.028 Mername-AA021 peptidase MER14055 MAP1D 254042 2q31.1 M24.950 Mername-AA020 peptidase homologue MER10972 12q11-q12 M24.973 proliferation-association protein 1 (proliferation-associated MER05497 PA2G4 5036 12q13 protein 1) M24.974 chromatin-specific transcription elongation factor 140 kDa MER26495 SUPT16H 11198 14q11.2 subunit M24.975 proliferation-associated protein 1-like (Homo sapiens MER29983 Xq23 chromosome X) M24.976 Mername AA-226 peptidase homologue (Homo sapiens) MER56262 442053 2q22.3 M24.977 Mername AA-227 peptidase homologue (Homo sapiens) MER47299 18q11.2-q12.1 (deduced from nucleotide sequence by MEROPS) MH M28 M28.010 glutamate carboxypeptidase II MER02104 FOLH1 2346 11p11.2 M28.011 NAALADASE L peptidase MER05239 NAALADL1 10004 11q12 M28.012 glutamate carboxypeptidase III MER05238 NAALAD2 10003 11q14.3-q21 M28.014 plasma glutamate carboxypeptidase (hematopoietic lineage MER05244 10404 8q22.2 switch 2) M28.016 Mername-AA103 peptidase MER15091 QPCTL 54814 19q13.32 M28.018 Fxna peptidase (Rattus norvegicus) (sequence assembled MER29965 KIAA1815 79956 9p24 by MEROPS) M28.972 transferrin receptor protein (transferrin receptor) MER02105 TFRC 7037 3q26.2 M28.973 transferrin receptor 2 protein (transferrin receptor 2) MER05152 TFR2 7036 7q22 M28.974 glutaminyl cyclase MER15095 QPCT 25797 2p22.3 M28.975 glutamate carboxypeptidase II (Homo sapiens)-like protein MER26971 NAALADL2 254827 3q26.31 M28.978 nicalin MER44627 NCLN 56926 19p13.3 MJ M38 M38.972 dihydro-orotase (dihydroorotase) MER05767 CAD 790 2p22-p21 M38.973 dihydropyrimidinase MER33266 DPYS 1807 8q22 M38.974 dihydropyrimidinase related protein-1 MER30143 CRMP1 1400 4p16.1-p15 M38.975 dihydropyrimidinase related protein-2 MER30155 DPYSL2 1808 8p22-p21 M38.976 dihydropyrimidinase related protein-3 MER30151 DPYSL3 1809 5q32 M38.977 dihydropyrimidinase related protein-4 MER30149 DPYSL4 10570 10q26 M38.978 dihydropyrimidinase related protein-5 MER30136 DPYSL5 56896 2p23.3 M38.979 hypothetical protein like 5730457F11RIK MER33184 51005 16p13.3 M38.980 1300019j08rik protein MER33186 144193 12q23.1 M38.981 guanine aminohydrolase MER37714 GDA 9615 9q21.11-21.33 MA M41 M41.004 i-AAA peptidase MER05755 YME1L1 10730 10p14 M41.006 paraplegin MER04454 SPG7 6687 16q24.3 M41.007 Afg3-like protein 2 MER05496 AFG3L2 10939 18p11 M41.010 Afg3-like protein 1 (deduced from nucleotide sequence by MER14306 AFG3L1 172 16q24 MEROPS) M41.011 Mername-AA024 peptidase MER01246 19 M43 M43.004 pappalysin-1 MER02217 PAPPA 5069 9q33.1 M43.005 pappalysin-2 MER14521 PAPPA2 60676 1q23-q25 M48 M48.003 farnesylated-protein converting enzyme 1 MER02646 ZMPSTE24 10269 1p34 M48.017 metalloprotease-related protein-1 MER30873 OMA1 115209 1p32 M- M49 M49.001 dipeptidyl-peptidase III MER04252 DPP3 10072 11q12-q13.1 M49.971 Mername-AA163 protein MER20074 9q21.31 M49.972 Mername-AA164 protein MER20410 4q13.1 MM M50 M50.001 S2P peptidase MER04458 MBTPS2 51360 X MP M67 M67.001 Poh1 peptidase MER20382 PSMD14 10213 2q24.3 M67.002 Jab1/MPN domain metalloenzyme MER22057 COPS5 10987 8q13.1 M67.003 Mername-AA165 peptidase MER21865 57559 10q23.31 M67.004 Mername-AA166 peptidase MER21890 CXorf53 79184 Xq28 M67.005 Mername-AA167 peptidase MER21887 MYSM1 114803 1p32.1 M67.006 AMSH deubiquitinating peptidase MER30146 STAMBP 10617 2p13.1 M67.008 putative peptidase (Homo sapiens chromosome 2) MER29970 2 M67.971 Mername-AA168 protein MER21886 EIF3S3 8667 8q24.11 M67.972 COP9 signalosome subunit 6 MER30137 COPS6 10980 7q22.1 M67.973 26S proteasome non-ATPase regulatory subunit 7 MER30134 PSMD7 5713 16q23-q24 M67.974 eukaryotic translation initiation factor 3 subunit 5 MER30133 EIF3S5 8665 11p15.4 M67.975 IFP38 peptidase homologue MER30132 EIF3S5 83880 11p15.4 M- M76 M76.001 Atp23 peptidase MER60642 PA S1 S01.010 granzyme B, human-type MER00168 GZMB 3002 14q11.2 S01.011 testisin MER05212 PRSS21 10942 16p13.3 S01.015 tryptase beta MER00137 TPSAB1 7177 16p13.3 tryptase beta (2) MER00136 TPSB2 64499 16p13.3 S01.017 kallikrein-related peptidase 5 MER05544 KLK5 25818 19q13.3-q13.4 S01.019 corin MER05881 CORIN 10699 4p13-p12 S01.020 kallikrein-related peptidase 12 MER06038 KLK12 43849 19q13.3-q13.4 S01.021 DESC1 peptidase MER06298 TMPRSS11E 28983 4q13.3 S01.028 tryptase gamma 1 MER11036 TPSG1 25823 16p13.3 S01.029 kallikrein-related peptidase 14 MER11038 KLK14 43847 19q13.3-q13.4 S01.033 hyaluronan-binding peptidase (HGF activator-like protein) MER03612 HABP2 3026 10q25.3 S01.034 transmembrane peptidase, serine 4 MER11104 TMPRSS4 56649 11q23.3 S01.047 adrenal secretory serine peptidase MER03734 TMPRSS11D 9407 4q13.2 S01.054 tryptase delta 1 (Homo sapiens) MER05948 TPSD1 23430 16p13.3 S01.072 matriptase-3 MER29902 TMPRSS7 344805 3q13 S01.074 marapsin MER06119 PRSS27 83886 16p13.3 S01.075 tryptase homologue 2 (Homo sapiens) MER06118 PRSS33 260429 16p13.3 S01.076 tryptase homologue 3 (Homo sapiens) MER00285 S01.079 transmembrane peptidase, serine 3 MER05926 TMPRSS3 64699 21q22.3 S01.081 kallikrein-related peptidase 15 MER00064 KLK15 55554 19q13.41 S01.085 Mername-AA031 peptidase MER14054 136541 7q34 S01.087 mosaic serine peptidase long-form MER14226 TMPRSS13 84000 11q23 S01.088 Mername-AA038 peptidase MER62848 138652 9q22.31 S01.098 Mername-AA128 peptidase (deduced from ESTs by MER16130 124221 16p13.3 MEROPS) S01.105 Mername-AA204 peptidase MER29980 S01.127 cationic trypsin (Homo sapiens-type) (1 (cationic)) MER00020 PRSS1 5644 7q35 S01.131 neutrophil elastase MER00118 ELA2 1991 19p13.3 S01.132 mannan-binding lectin-associated serine peptidase-3 MER31968 MASP1 5648 3q27-q28 S01.133 cathepsin G MER00082 CTSG 1511 14q11.2 S01.134 myeloblastin (proteinase 3) MER00170 PRTN3 5657 19p13.3 S01.135 granzyme A MER01379 GZMA 3001 5q11-q12 S01.139 granzyme M MER01541 GZMM 3004 19p13.3 S01.140 chymase (human-type) MER00123 CMA1 1215 14q11.2 S01.143 tryptase alpha (1) MER00135 TPSAB1 7176 16p13.3 S01.146 granzyme K MER01936 GZMK 3003 5q11-q12 S01.147 granzyme H MER00166 GZMH 2999 14q11.2 S01.152 chymotrypsin B MER00001 CTRB1 1504 16q23.2-q23.3 S01.153 pancreatic elastase MER03733 ELA1 1990 12q13 S01.154 pancreatic endopeptidase E (A) MER00149 ELA3A 10136 1p36.12 S01.155 pancreatic elastase II (IIA) MER00146 63036 1p36.21 S01.156 enteropeptidase MER02068 PRSS7 5651 21q21 S01.157 chymotrypsin C MER00761 CTRC 11330 1p36.21 S01.159 prostasin MER02460 PRSS8 5652 16p11.2 S01.160 kallikrein hK1 MER00093 KLK1 3816 19q13.2-q13.4 S01.161 kallikrein-related peptidase 2 MER00094 KLK2 3817 19q13.2-q13.4 S01.162 kallikrein-related peptidase 3 MER00115 KLK3 354 19q13.3-q13.4 S01.174 mesotrypsin MER00022 PRSS3 5646 9p13 S01.189 complement component C1r-like peptidase MER16352 C1RL 51279 12p13.31 S01.191 complement factor D MER00130 DF 1675 19 S01.192 complement component activated C1r MER00238 C1R 715 12p13 S01.193 complement component activated C1s MER00239 C1S 716 12p13 S01.194 complement component C2a MER00231 C2 717 6p21.3 S01.196 complement factor B MER00229 BF 629 6p21.3 S01.198 mannan-binding lectin-associated serine peptidase 1 MER00244 MASP1 5648 3q27-q28 S01.199 complement factor I MER00228 IF 3426 4q24-q25 S01.205 pancreatic endopeptidase E form B (B) MER00150 ELA3B 23436 1p36.12 S01.206 pancreatic elastase II form B (Homo sapiens) (IIB) MER00147 ELA1 51032 12q13 S01.211 coagulation factor XIIa MER00187 F12 2161 5q33-qter S01.212 plasma kallikrein MER00203 KLKB1 3818 4q35 S01.213 coagulation factor XIa MER00210 F11 2160 4q35 S01.214 coagulation factor IXa MER00216 F9 2158 Xq27.1-q27.2 S01.215 coagulation factor VIIa MER00215 F7 2155 13q34 S01.216 coagulation factor Xa MER00212 F10 2159 13q34 S01.217 thrombin MER00188 F2 2147 11p11-q12 S01.218 protein C (activated) MER00222 PROC 5624 2q13-q14 S01.223 acrosin MER00078 ACR 49 22q13-qter S01.224 hepsin MER00156 HPN 3249 19q11-q13.2 S01.228 hepatocyte growth factor activator MER00186 HGFAC 3083 4p16 S01.229 mannan-binding lectin-associated serine peptidase 2 MER02758 MASP2 10747 1p36.3-p36.2 S01.231 u-plasminogen activator MER00195 PLAU 5328 10q24 S01.232 t-plasminogen activator MER00192 PLAT 5327 8p12 S01.233 plasmin MER00175 PLG 5340 6q26 S01.236 kallikrein-related peptidase 6 (Homo sapiens) MER02580 KLK6 5653 19q13.3-q13.4 S01.237 neurotrypsin MER04171 PRSS12 8492 4q25-q26 S01.244 kallikrein-related peptidase 8 MER05400 KLK8 11202 19q13.3-q13.4 S01.246 kallikrein-related peptidase 10 MER03645 KLK10 5655 19q13.33 S01.247 epitheliasin MER03736 TMPRSS2 7113 21q22.3 S01.251 kallikrein-related peptidase 4 MER05266 KLK4 9622 19q13.3-q13.4 S01.252 prosemin MER04214 PRSS22 64063 16p13.3 S01.256 chymopasin MER01503 CTRL 1506 16q22.1 S01.257 kallikrein-related peptidase 11 MER04861 KLK11 11012 19q13.3-q13.4 S01.258 trypsin-2 (human-type) (II) MER00021 PRSS2 5645 7q35 S01.277 HtrA1 peptidase MER02577 PRSS11 5654 10q25.3-q26.2 S01.278 HtrA2 peptidase MER04093 PRSS25 27429 2p12 S01.284 HtrA3 peptidase MER14795 HTRA3 94031 4p16.1 S01.285 HtrA4 peptidase MER16351 HTRA4 203100 8p11.23 S01.286 Tysnd1 peptidase MER50461 TYSND1 219743 10q22.1 S01.291 LOC144757 peptidase (Homo sapiens) and similar (protein MER17085 TMPRSS12 283471 12q13.13 sequence extended by use of MEROPS EST alignment) S01.292 HAT-like putative peptidase 2 MER21884 TMPRSS11A 339967 4q13.3 S01.298 trypsin C MER21898 154754 7q34 S01.299 Mername-AA175 peptidase MER21930 203074 8p23.1 S01.300 kallikrein-related peptidase 7 MER02001 KLK7 5650 19q13.3-q13.4 S01.302 matriptase MER03735 ST14 6768 11q24-q25 S01.306 kallikrein-related peptidase 13 MER05269 KLK13 26085 19q19.3-q19.4 S01.307 kallikrein-related peptidase 9 MER05270 KLK9 23579 19q19.3-q19.4 S01.308 matriptase-2 MER05278 TMPRSS6 164656 22q13.1 S01.309 umbelical vein peptidase MER05421 PRSS23 11098 11q14.1 S01.311 LCLP peptidase (LCLP (N-terminus)) MER01900 S01.313 spinesin MER14385 TMPRSS5 80975 11q23.3 S01.318 marapsin-2 MER21929 339501 1q42.13 S01.319 complement factor D-like putative peptidase MER56164 PRSSL1 400668 19p13.3 S01.320 Mername-AA180 peptidase MER22410 OVCH2 341277 11p15.4 S01.321 Mername-AA181 peptidase MER44589 TMPRSS11F 389208 4q13.2 S01.322 Mername-AA182 peptidase MER22412 OVCH1 341350 12p11.23 S01.325 epidermis-specific SP-like putative peptidase MER29900 345062 4q31.3 S01.326 testis serine peptidase 5 MER29901 377047 3p21 S01.327 testis serine peptidase 1 MER30190 360226 16p13.3 S01.357 polyserase-IA (unit 1) (unit 1) MER30879 TMPRSS9 360200 19p13.3 S01.358 polyserase-IA (unit 2) (unit 2) MER30880 TMPRSS9 360200 19p13.3 S01.362 testis serine peptidase 2 (human-type) MER33187 339906 3p21.31 S01.363 hypothetical acrosin-like peptidase (Homo sapiens) MER33253 284967 2q14.1 S01.365 Mername-AA221 putative peptidase MER28215 TMPRSS11B 132724 4q13.3 S01.374 polyserase-3 (unit 1) MER61763 S01.375 polyserase-3 (unit 2) MER61748 S01.376 peptidase similar to tryptophan/serine protease MER56263 346702 8p23.1 S01.414 polyserase-2 (unit 1) MER61777 S01.940 polyserase-2 (unit 2) MER61760 S01.941 polyserase-2 (unit 3) MER65694 S01.957 secreted trypsin-like serine peptidase homologue (deduced MER30000 4 from nucleotide sequence by MEROPS) S01.969 polyserase-1A (unit 3) (unit 3) MER29880 TMPRSS9 360200 19p13.3 S01.971 azurocidin (azurocidin) MER00119 AZU1 566 19p13.3 S01.972 haptoglobin-1 (haptoglobin-2) MER00233 HP 3240 16q22.1 S01.974 haptoglobin-related protein (haptoglobin-related protein) MER00235 HPR 3250 16q22.1 S01.975 macrophage-stimulating protein (macrophage-stimulating MER01546 MST1 4485 3p21 protein) S01.976 hepatocyte growth factor (hepatocyte growth factor) MER00185 HGF 3082 7q21.1 S01.977 hepatocyte growth factor-like protein homologue MER03611 MST1 4485 3p21 (hepatocyte growth factor-like protein homologue) S01.979 protein Z (protein Z) MER00227 PROZ 8858 13q34 S01.985 TESP1 protein (deduced from nucleotide sequence by MER47214 646743/ 2q21.1 MEROPS) 646747 S01.989 LOC136242 gene product (protein sequence amended by MER16132 7q34 use of MEROPS EST alignment) S01.992 Mername-AA199 MER16346 221191 16q21 S01.993 testis-specific protein TSP50 MER16347 29122 3p14-p12 S01.994 dj223e3.1 protein (Homo sapiens) MER16350 PRSS35 167681 6q15 S01.998 DKFZp586H2123-like protein MER66474 S01.999 apolipoprotein MER00183 LPA 4018 6q27 S01.P08 psi-KLK1 pseudogene (Homo sapiens) MER33287 KLKP1 19q13.41 S01.P09 tryptase pseudogene I MER15077 16p13.3 S01.P10 tryptase pseudogene II MER15078 16p13.3 S01.P11 tryptase pseudogene III MER15079 16p13.3 SB S8 S08.011 kexin-like peptidase (Pneumocystis carinii) (MEROPS MER62850 651834 assumes this sequence to be derived from a contamination by Pneumocystis carinii) S08.039 proprotein convertase 9 MER22416 PCSK9 255738 1p32.2 S08.063 site-1 peptidase (KIAA0091 protein) MER01948 MBTPS1 8720 16q24 S08.071 furin MER00375 FURIN 5045 15q25-q26 S08.072 proprotein convertase 1 MER00376 PCSK1 5122 5q15-q21 S08.073 proprotein convertase 2 MER00377 PCSK2 5126 20p11.2 S08.074 proprotein convertase 4 MER28255 PCSK4 54760 19p13.3 S08.075 PACE4 proprotein convertase MER00383 PCSK6 5046 15q26 S08.076 proprotein convertase 5 MER02578 PCSK5 5125 9 S08.077 proprotein convertase 7 MER02984 PCSK7 9159 11q23-q24 S08.090 tripeptidyl-peptidase II MER00355 TPP2 7174 13q32-q33 SC S9 S09.001 prolyl oligopeptidase MER00393 PREP 5550 6q22 S09.003 dipeptidyl-peptidase IV (eukaryote) MER00401 DPP4 1803 2q23-qter S09.004 acylaminoacyl-peptidase MER00408 APEH 327 3p21 S09.007 fibroblast activation protein alpha subunit MER00399 FAP 2191 2q23 S09.015 PREPL A protein MER04227 PREPL 9581 2 S09.018 dipeptidyl-peptidase 8 MER13484 DPP8 54878 15q22 S09.019 dipeptidyl-peptidase 9 (R26984_1 protein) MER04923 DPP9 91039 19p13.3 S09.051 FLJ1 putative peptidase MER17240 C13orf6 84945 13q33.3 S09.052 Mername-AA194 putative peptidase MER17353 C19orf27 81926 19p13.3 S09.053 Mername-AA195 putative peptidase MER17367 58489 15q25.1 S09.054 Mername-AA196 putative peptidase MER17368 C20orf22 26090 20p11.1 S09.055 Mername-AA197 putative peptidase MER17371 C9orf77 51104 9q21.12 S09.061 C14orf29 protein MER33244 C14orf29 145447 14q22.1 S09.062 hypothetical protein MER33245 ABHD10 55347 3q13.2 S09.063 hypothetical esterase/lipase/thioesterase (deduced from MER47309 3 nucleotide sequence by MEROPS) S09.065 protein bat5 MER37840 BAT5 7920 6p21.3 S09.958 hypothetical protein flj40219 MER33212 79984 16q22.1 S09.959 hypothetical protein flj37464 MER33240 283848 16q22.1 S09.960 hypothetical protein flj33678 MER33241 221223 16q12.2 S09.966 hypothetical protein flj90714 (Homo sapiens) MER37720 C13orf6 84945 13q33.3 S09.973 dipeptidylpeptidase homologue DPP6 (DPP6 protein) MER00403 DPP6 1804 7 S09.974 dipeptidylpeptidase homologue DPP10 MER05988 DPP10 57628 2q12.3-2q14.2 S09.976 protein similar to chromosome 20 open reading frame 135 MER37845 C20orf135 140701 20q13.33 (Mus musculus) S09.977 kynurenine formamidase MER46020 AFMID 125061 17q25.3 S09.978 thyroglobulin precursor (thyroglobulin) MER11604 TG 7038 8q24.2-q24.3 S09.979 acetylcholinesterase MER33188 ACHE 43 7q22 S09.980 cholinesterase MER33198 BCHE 590 3q26.1-q26.2 S09.981 carboxylesterase D1 MER33213 S09.982 liver carboxylesterase MER33220 CES1 1066 16q13-q22.1 S09.983 carboxylesterase 3 MER33224 CES3 23491 S09.984 carboxylesterase 2 MER33226 CES2 8824 16q22.1 S09.985 bile salt-dependent lipase MER33227 CEL 1056 9q34.3 S09.986 carboxylesterase-related protein MER33231 CES4 51716 16q13 S09.987 neuroligin 3 MER33232 NLGN3 54413 Xq13.1 S09.988 neuroligin 4, X-linked MER33235 NLGN4X 57502 Xp22.33 S09.989 neuroligin 4, Y-linked MER33236 NLGN4Y 22829 Yq11.221 S09.990 esterase D (Homo sapiens) MER43126 ESD 2098 13q14.1-q14.2 S09.991 arylacetamide deacetylase MER33237 AADAC 13 3q21.3-q25.2 S09.992 KIAA1363-like protein MER33242 AADACL1 57552 3q26.31 S09.993 hormone-sensitive lipase MER33274 LIPE 3991 19q13.2 S09.994 neuroligin 1 MER33280 NLGN1 22871 3q26.32 S09.995 neuroligin 2 MER33283 NLGN2 57555 17q13.2 S10 S10.002 serine carboxypeptidase A MER00430 PPGB 5476 20q13.1 S10.003 vitellogenic carboxypeptidase-like protein MER05492 CPVL 54504 7p14-p15.3 (WUGSC:H_RG113D17.1 protein) S10.013 RISC peptidase MER10960 SCPEP1 59342 17 SE S12 S12.004 LACT-1 peptidase MER17071 LACTB 114294 15q22.1 SK S14 S14.003 peptidase Clp (type 3) MER02211 CLPP 8192 19 SJ S16 S16.002 PIM1 peptidase MER00495 PRSS15 9361 19p13.2 S16.006 Mername-AA102 peptidase MER14970 83752 16q12.1 SF S26 S26.009 signalase (eukaryote) 18 kDa component (18 kDa) MER05386 SEC11L1 23478 15q25.2 S26.010 signalase (eukaryote) 21 kDa component MER14880 SEC11L3 90701 18q21.31 S26.012 mitochondrial inner membrane peptidase 2 MER14877 IMMP2L 83943 7q31 S26.013 mitochondrial signal peptidase (metazoa) MER13949 196294 11p13 S26.022 Mername AA-228 putative peptidase (Homo sapiens) MER47379 8 (deduced from nucleotide sequence by MEROPS) SC S28 S28.001 lysosomal Pro-Xaa carboxypeptidase MER00446 PRCP 5547 11q14 S28.002 dipeptidyl-peptidase II MER04952 DPP7 29952 9q34.3 S28.003 thymus-specific serine peptidase MER05538 PRSS16 10279 6p21.31-p22.2 S33 S33.011 epoxide hydrolase-like putative peptidase MER31614 ABHD8 79575 19p13.12 S33.012 Loc328574-like protein MER33246 SERHL 253190 22q13.2-q13.31 S33.013 abhydrolase domain-containing protein 4 MER31616 ABHD4 63874 14q11.2 S33.971 epoxide hydrolase (epoxide hydrolase) MER00432 EPHX1 2052 1q42.1 S33.972 mesoderm specific transcript protein MER17123 MEST 4232 7q32 S33.973 cytosolic epoxide hydrolase MER29997 EPHX2 2053 8p21-p12 S33.974 similar to hypothetical protein FLJ22408 MER31608 ABHD7 253152 1p22.1 S33.975 CGI-58 putative peptidase MER30163 ABHD5 51099 3p25.3-p24.3 S33.976 Williams-Beuren syndrome critical region protein 21 MER31610 ABHD11 83451 7q11.23 epoxide hydrolase S33.977 epoxide hydrolase MER31612 ABHD6 57406 3p21.2 S33.978 hypothetical protein fli22408 (epoxide hydrolase) (Homo MER31617 ABHD9 79852 19p13.13 sapiens) S33.980 monoglyceride lipase MER33247 MGLL 11343 3q21.3 S33.981 hypothetical protein MER33249 ABHD14A 25864 3p21.1 S33.982 valacyclovir hydrolase MER33259 BPHL 670 6p25 S33.983 Ccg1-interacting factor b MER33263 84836 3p21.31 S33.984 protein phosphatase methylesterase 1 MER37853 51400 11q13.4 S33.986 NDRG4 protein MER42913 NDRG4 65009 16q21-q22.1 S33.987 NDRG3 protein MER42914 NDRG3 57446 20q11.21-q11.23 S33.988 Mername AA-229 peptidase homologue (Homo sapiens) MER45809 NDRG1 10397 8q24.3 SK S41 S41.950 interphotoreceptor retinoid-binding protein, unit 1 MER30235 RBP3 5949 10q11.2 S41.951 interphotoreceptor retinoid-binding protein, unit 2 MER59675 RBP3 5949 10q11.2 SB S53 S53.003 tripeptidyl-peptidase I MER03575 TPP1 1200 11p15 ST S54 S54.002 rhomboid-like protein 2 MER15453 RHBDL2 54933 1p35.1 S54.005 rhomboid-like protein 1 MER15454 RHBDL1 9028 16p13.3 S54.006 ventrhoid transmembrane protein MER20285 RHBDL4 162494 17q11.2 S54.008 rhomboid-like protein 5 MER30173 84236 2q36.3 S54.009 Rhomboid-7 (Drosophila melanogaster) MER30047 PSARL 55486 3q27.3 S54.952 RHBDF1 protein MER04528 RHBDF1 64285 16pter-p13 S54.953 peptidase homologue similar to hypothetical protein MER02969 RHBDL6 79651 17q25.3 FLJ22341 S54.955 rhomboid-like protein 7 MER31620 RHBDL7 57414 7q11.23 SP S59 S59.001 nucleoporin 145 MER20203 NUP98 4928 11p15.5 S59.951 nup 36 protein (Homo sapiens) and similar MER20219 SR S60 S60.001 lactoferrin (unit 1) MER20365 LTF 4057 3q21-q23 S60.970 lactotransferrin precursor, domain 2 (unit 2) MER37758 LTF 4057 3q21-q23 S60.972 serotransferrin precursor (domain 1) (unit 1) MER33288 TF 7018 3q22.1 S60.973 melanotransferrin domain 1 (unit 1) MER33291 MFI2 4241 3q28-q29 S60.975 serotransferrin precursor (domain 2) (unit 2) MER37088 TF 7018 3q22.1 S60.976 melanotransferrin domain 2 (unit 2) MER37142 MFI2 4241 3q28-q29 S— S63 S63.001 EGF-like module containing mucin-like hormone receptor- MER37230 EMR2 30817 19p13.1 like 2 S63.002 CD97 antigen MER37286 CD97 976 19p13 S63.003 EGF-like module containing mucin-like hormone receptor- MER37288 EMR3 84658 19p13.1 like 3 S63.004 EGF-like module containing mucin-like hormone receptor- MER37278 EMR1 37278 19p13.3 like 1 (Homo sapiens) S63.008 EGF-like module containing mucin-ike hormone receptor- MER37294 EMR4 326342 19p13.3 like 4 S63.009 cadherin EGF LAG seven-pass G-type receptor 2 precursor MER45397 CELSR2 1952 1p21 (Homo sapiens) S68 S68.001 PIDD auto-processing protein unit 1 MER20001 11p15.5 S68.002 PIDD auto-processing protein unit 2 MER63690 11p15.5 PB T1 T01.010 proteasome catalytic subunit 1 MER00556 PSMB6 5694 17p13 T01.011 proteasome catalytic subunit 2 MER02625 PSMB7 5695 9q34.11-q34.12 T01.012 proteasome catalytic subunit 3 MER02149 PSMB5 5693 14q11.2 T01.013 proteasome catalytic subunit 1i MER00552 PSMB9 5698 6p21.3 T01.014 proteasome catalytic subunit 2i MER01515 PSMB10 5699 16q22.1 T01.015 proteasome catalytic subunit 3i MER00555 PSMB8 5696 6p21.3 T01.016 RIKEN cDNA 5830406J20 MER26203 122706 14q11.2 T01.017 protein serine kinase c17 (Homo sapiens) MER26497 T01.971 proteasome subunit alpha 6 MER00557 PSMA6 5687 14q13 T01.972 proteasome subunit alpha 2 MER00550 PSMA2 5683 6q27 T01.973 proteasome subunit alpha 4 MER00554 PSMA4 5685 15q11.2 T01.974 proteasome subunit alpha 7 (XAPC7) MER04372 PSMA7 5688 20pter-p12.1 proteasome subunit alpha 7 MER91448 T01.975 proteasome subunit alpha 5 MER00558 PSMA5 5686 1p13 T01.976 proteasome subunit alpha 1 MER00549 PSMA1 5682 11p15.1 T01.977 proteasome subunit alpha 3 MER00553 PSMA3 5684 14q23 T01.978 2410072d24rik protein (mouse) MER33250 PSMA8 143471 18q11.2 T01.983 proteasome subunit beta 3 MER01710 PSMB3 5691 2q35 T01.984 proteasome subunit beta 2 MER02676 PSMB2 5690 1p34.2 T01.986 proteasome subunit beta 1 MER00551 PSMB1 5689 7p12-p13 proteasome subunit beta 1 MER91422 T01.987 proteasome subunit beta 4 MER01711 PSMB4 5692 1q21 T01.991 Mername AA-230 peptidase homologue (Homo sapiens) MER47329 2q33 (deduced from nucleotide sequence by MEROPS) T01.P02 Mername AA-231 pseudogene (Homo sapiens) (deduced MER47172 PSMB3P 121131 12q13.2 from nucleotide sequence by MEROPS) T01.P03 Mername AA-232 pseudogene (Homo sapiens) (deduced MER47316 130700 2q35 from nucleotide sequence by MEROPS) T2 T02.001 glycosylasparaginase precursor MER03299 AGA 175 4q23-q27 T02.002 isoaspartyl dipeptidase (threonine type) MER31622 ASRGL1 80150 11q12.3 T02.004 taspase-1 MER16969 TASP1 55617 20p12.1 T3 T03.002 gamma-glutamyltransferase 5 (mammalian) (5) MER01977 GGTLA1 2687 22q11.23 T03.006 gamma-glutamyltransferase 1 (mammalian) (1) MER01629 GGT1 2678 22q11.23 T03.015 gamma-glutamyltransferase 2 (Homo sapiens) (2) MER01976 GGT2 2679 22q11.23 T03.016 gamma-glutamyltransferase-like protein 4 (m-type 3) MER02721 GGTL4 91227 22q11.21 T03.017 gamma-glutamyltransferase-like protein 3 MER16970 GGTL3 2686 20q11.22 T03.018 similar to gamma-glutamyltransferase 1 precursor (Homo MER26204 22q11.21 sapiens) T03.019 similar to gamma-glutamyltransferase 1 precursor (Homo MER26205 22q11.23 sapiens) T03.021 Mername-AA211 putative peptidase MER26207 22 T03.971 gamma-glutamyl transpeptidase homologue MER37241 2p11.1 (chromosome 2, Homo sapiens) U- U48 U48.002 prenyl peptidase 1 (protein sequence corrected by use of MER04246 RCE1 9986 11q13 MEROPS EST alignment)

Retroviral Proteases

Recombinant human retroviral proteases nay also be used for the present invention. Human retroviral proteases, including that of human inmmunodeficiency virus type 1 (HIV-1) (Beck et al., 2002), human T cell leukemia viruses (HTLV) (Shuker et al., Chem. Biol. 10:373 (2003)), and severe acute respiratory syndrome coronavirus (SARS), have been extensively studied as targets of anti-viral therapy. These proteases often have long recognition sequences and high substrate selectivity. For example, SQNY↓PIV (SEQ ID NO:60) was determined as a preferred cleavage sequence of HIV-1 protease (Beck et al. Curr. Drug Targets Infect. Disord. 2(1):37-50 (2002), the preferred cleavage sequence for HTLV protease has been determined to be PVIL↓PIQA (SEQ ID NO:61) (Naka et al. Bioorg. Med. Chem. Lett. 16(14):3761-3764 (2006).

Coronaviral Proteases

Coronaviral or toroviral proteases are encoded by members of the animal virus family Coronaviridae and exhibit high cleavage specificity. Such proteases are another preferred embodiment for the present invention. The SARS 3C-like protease has been found to selectively cleave at AVLQ↓SGF (SEQ ID NO:62) (Fan et al. Biochem. Biophys. Res. Commun. 329(3):934-940 (2005)).

Picornaviral Proteases

Picornaviral proteases may also be used for the present invention. Such picornaviral proteases have been studied as targets of anti-viral therapy, for example human Rhinovirus (HRV) (Binford et al., Antimicrob. Agents Chemother. 49:619 (2005)). HRV 3C protease recognizes and cleaves ALFQ↓GP (SEQ ID NO:63) (Cordingley et al. J. Biol. Chem. 265(16):9062-9065 (1990)).

Potyviral Proteases

Potyviral proteases are encoded by members of the plant virus family Potyviridae and exhibiting high cleavage specificity, and are another preferred embodiment for the present invention. For example, tobacco etch virus (TEV) protease has very high substrate specificity and catalytic efficiency, and is used widely as a tool to remove peptide tags from overexpressed recombinant proteins (Nunn et al., J. Mol. Biol. 350:145 (2005)). TEV protease recognizes an extended seven amino acid residue long consensus sequence E-X-X-Y-X-Q↓S/G (where X is any residue) that is present at protein junctions (SEQ ID NO:59). Those skilled in the art would recognize that it is possible to engineer a particular protease such that its sequence specificity is altered to prefer another substrate sequence (Tozser et al., FEBS J. 272:514 (2005)).

Proteases of Other Origins

Since proteases are physiologically necessary for living organisms, they are ubiquitous, being found in a wide range of sources such as plants, animals, and microorganisms (Rao et al. Microbiol. Mol. Biol. Rev. 62(3):597-635 (1998)). All these proteases are potential candidates for the present invention. In a preferred embodiment, PEGylation may be utilized to reduce the immunological potential of fusion proteases for the present invention, particularly for those that are of non-human origins. PEGylation may confer additional benefits to protease fusion proteins, such as improved plasma persistence and reduced non-specific cell binding.

B. Recombinant DNA Construct Design and Sequence Modifications

Methods described above for the construction and sequence modification of fusion proteins, such as DT fusion proteins, are generally applicable to construction of protease fusion proteins as well, except for those techniques specifically dedicated to diphtheria toxin. Many proteases found in nature are synthesized as zymogens, i.e., as catalytically inactive forms in which an inhibitory peptide binds to and masks the active site, or in which the active site is otherwise nonfunctional because the presence of an inhibitory peptide alters the conformation of the active site. Zymogens are typically activated by cleavage and release of the inhibitory peptide. In one embodiment of the present invention, the exogenous protease of the protoxin activator is in the form of a zymogen, which may be activated by another exogenous protease or by an endogenous protease. Depending on the location of the inhibitory peptide in the primary sequence, such zymogens are either favorably N-terminally situated (when the inhibitory peptide is located at the N-terminus of the zymogen) or C-terminally situated (when the inhibitory peptide is located at the C-terminus of the zymogen). When the protease moiety of the protoxin activator is linked to the cell-targeting moiety by chemical or enzymatic linkage, the inhibitory peptide may be located at either the N-terminus or the C-terminus, since either or both termini may be free as a result of an operable linkage to a cell-targeting moiety taking place at a location other than the N- or C-terminus.

Accordingly, one embodiment of the present invention comprises a recombinant protoxin proactivator that may be activated by another protease. Such a protoxin proactivator comprises an inhibitory peptide, a modifiable activation moiety, a protease moiety, and a cell-targeting moiety. The inhibitory peptide is removed by a modification of the modifiable activation moiety that either directly or indirectly cleaves the modifiable activation moiety to afford an active protease fusion.

Many zymogens comprise active enzymatic moieties in which the inhibitory peptide physically occupies the active site substrate binding cleft, and for which the cleavage site that releases the inhibitory peptide lies distal to the cleft. Among members of a class of proteases for which the active site is composed of residues at the N-terminus of the polypeptide chain, and for which the alpha amino group comprises the active site nucleophile or an important determinant of catalytic efficacy, artificial zymogens can be formed by directly appending a protease cleavage site to the N-terminus. In such cases the activating protease must be capable of cleaving the bond between the recognition site and the desired N-terminal residue. In a preferred embodiment, the activating protease has no sequence requirement for the residue directly following the cleavage location, or preferentially cleaves substrates for which the residue directly following the cleavage location is the same as the reside at the N-terminus of the mature protease. Examples of activating proteases that directly cleave the modifiable activation moiety and their corresponding cleavage sites include, but are not limited to, IEGR↓, a protease cleavage site targeted by Factor Xa; DDDDK↓, (SEQ ID NO:25), a protease cleavage site targeted by enterokinase. Specifically, a GrB fusion containing DDDDK (SEQ ID NO:25), to its N-terminus may be generated and activated by treatment with enterokinase. Specifically, GrB-anti-CD19, GrB-anti-CD5, and GrB-(YSA)₂ fusions are so constructed.

In another embodiment of the present invention, the proactivator may be activated in vivo by a proteolytic activity that is endogenous to the targeted cells. One example of such endogenous protease is furin, an endosomal protease that is ubiquitously expressed in various mammalian cells. Specifically, a furin recognition site such as RVRR↓ (SEQ ID NO:64) may replace a natural zymogen cleavage site to provide a zymogen that is activated by proximity to the cell surface or by internalization. In the case of proteases for which the N-terminal residues comprise important determinants of the active site, such a furin recognition site can be directly appended to the N-terminus of the proactivator. For example, a furin cleavage site can be added to the N-terminus of Granzyme B or Granzyme M to provide an natively activatable proactivator. Specifically, a GrB fusion construct containing two C-terminal 12 residue cell-targeting YSA peptides and an N-terminal furin cleavage site is prepared for the production of GrB-(YSA)₂ (FIG. 20).

Protoxin proactivators containing a furin cleavage site are preferably produced in expression systems that do not contain native furin activity, e.g., in E. coli. A protoxin proactivator that is activatable in the targeted human cells by intracellular furin during its internalization process is an example of a natively-activatable protoxin proactivator. One important advantage of such a protoxin proactivator, as compared to a protoxin activator, is that the protoxin proactivator may be combined with a protoxin for simplified therapeutic delivery. Such mixtures of protoxins and protoxin proactivators will show reduced activation prior to accumulation upon the targeted cells.

Protoxin proactivator proteins that are activated by proteolytic cleavage by an endogenous protease activity of the target cell can be designed so that the proteolytic cleavage severs the operable linkage between the cell-targeting moiety and the catalytic or activator moiety. For example in a translational fusion, the inhibitory peptide might lie between the cell-targeting moiety and the catalytic moiety. Or in a chemically or enzymatically induced crosslinking of cell-targeting moiety to catalytic or activator moiety, the crosslinking may be induced via residues on the inhibitory peptide moiety that are not functionally required for inhibition of the catalytic or activator moiety.

Strategies to Reduce Potential Side Effects of Protease Fusions

Application of human proteases for immunotoxin activation may encounter complications if the protease of choice is capable of eliciting unintended biological effects in addition to the designed toxin activation. For example, many proteases, including granzymes and caspases, can promote cell death through involvement in an apoptotic cascade. Immunotoxins composed of granzyme B and a cell surface targeting domain have been developed as cytotoxic agents against certain diseased cell populations (Liu et al. Neoplasia 8:125-135 (2006), Dalken et al. Cell Death Differ. 13:576-585, Zhao et al. J. Biol. Chem. 279:21343-21348 (2004), U.S. Pat. No. 0,710,1977). To eliminate such potential side effects in the context of the present invention, it is preferable to use a cell surface target that does not internalize upon binding as the intended target for the protease fusion protein. In such a case the protoxin activation may be accomplished on the cell surface, but a toxic effect will not be generated by the protoxin activator acting alone.

Another approach is to mutate the candidate proteases so that they confer altered sequence specificity, thus are no longer preferentially bound to and cleaving at the native cleavage sites. Such engineered proteases are likely to have lower toxicities that are caused by biological cascade downstream from the proteolytic processing at the naturally occurring cleavage sequence. Selection or screening methods that are suited for such applications have been developed (e.g., Sices et al. Proc. Natl. Acad. Sci. USA 95:2828-2833 (1998) and Baum et al. Proc. Natl. Acad. Sci. USA 87:10023-10027 (1990)), and have been used select mutant proteases that are capable of cleaving a sequence that is different from the native proteolytic site of the original protease (e.g., O'Loughlin et al. Mol. Biol. Evol. 23:764-722 (2006), Han et al. Biochem. Biophy. Res. Commun. 337:1102-1106 (2005), and Venekei et al. Protein Eng. 9:85-93 (1996)). Because the cleavage site and the inhibitor RCL often possess sequence similarity, changing the proteolytic specificity of a protease may also result in its resistance to inhibition by its known proteinase inhibitors. Examples are available where the selection or screening for altered cleavage site, lower cytotoxicity, and altered inhibition profile are accomplished simultaneously (O'Loughlin et al. Mol. Biol. Evol. 23:764-722 (2006)). Specifically, granzyme B is modified to provide altered forms of granzyme with reduced spontaneous toxicity through altered substrate specificity.

Further modifications can be engineered to increase the activity and/or specificity of proteases. These modifications include PEGylation to increase stability to serum or to lower immunogenicity, and genetic engineering/selection may produce mutant proteases that possess altered properties such as resistance to certain inhibitors, increased thermal stability, and improved solubility.

Strategies to Prevent Inhibition by Proteinase Inhibitors in Plasma and in Cells

In designing and utilizing protease fusions of the invention, it should be noted that proteinase inhibitors may hamper the proteolytic activities of protease fusion proteins. For example, GrB is specifically inhibited by intracellular proteinase inhibitor 9 (PI-9), a member of the serpin superfamily that primarily exists in cytotoxic lymphocytes (Sun et al., J. Biol. Chem. 271:27802 (1996)) and has been detected in human plasma. GrB can also be inhibited by α₁-protease inhibitor (α₁PI) that is present in human plasma (Poe et al., J. Biol. Chem. 266:98(1991)). GrM is inhibited by α₁-antichymotrypsin (ACT) and α₁PI (Mahrus et al., J. Biol. Chem. 279:54275 (2004)), and GrA is inhibited in vitro by protease inhibitors antithrombin III (ATIII) and α₂-macroglobulin (α₂M) (Spaeny-Dekking et al., Blood 95:1465 (2000)). These proteinase inhibitors are also present in human plasma (Travis and Salvesen, Annu. Rev. Biochem. 52:655 (1983)).

One approach to preserve proteolytic activities of granzymes is to utilize complexation with proteoglycan, since the mature and active form of GrA has been observed in human plasma as a complex with serglycin, a granule-associated proteoglycan (Spaeny-Dekking et al., Blood 95:1465 (2000)). Glycosaminglycan complexes of GrB have also been found proteolytically active (Galvin et al., J. Immunol. 162:5345 (1999)). Thus, it may be possible to keep granzyme fusion proteins active in plasma through formulations using chondroitin sulfates.

Alternatively, potential candidate proteases may be screened in vitro by interactions with known proteinase inhibitors in plasma or with human plasma directly to avoid potential complications posed by these proteinase inhibitors. Alternatively, proteases for which cognate inhibitors are found in plasma can be engineered to provide mutant forms that resist inhibition. For example, in vitro E. coli expression-screening methods have been developed to select mutant proteases that are resistant to known HIV-1 protease inhibitors (Melnick et al., Antimicrob. Agents Chemother. 42:3256 (1998)).

C. Expression of Protease Fusion Proteins

Methods for the overexpression of large fusion proteins are well known in the art and can be applied to the overexpression of the protease fusion proteins of the invention. Examples of expression systems that may be used in the construction of the fusion proteins of the invention are E. coli, baculovirus in insect cells, yeast systems in Saccharomyces cerevisiae and Pichia pastoris, mammalian cells, and transient expression in vaccinia. Methods described above for the expression of DT fusion proteins are generally applicable for protease fusion proteins, except for those solely applicable to diphtheria toxin.

A mammalian expression system can be used to produce the protease fusion protein, particularly when a protease of human origin such as human granzyme B is selected as the protease portion of the fusion. Expressing proteases of human origin in mammalian cells has certain advantages, notably providing glycosylation patterns that are identical to or closely resemble native forms, which are not immunogenic and may help the folding, solubility, and stability of the recombinant protein.

PEGylation of Proteins

One embodiment of the present invention is the utilization of PEGylated fusion proteins. Preferred embodiments are site-specifically PEGylated fusion proteins. It is known in the art that PEGylated proteins can exhibit a broad range of bioactivities due to the site, number, size, and type of PEG attachment (Harris and Chess Nat. Rev. Drug Discov. 2(3):214-221 (2003)). A preferred composition of a fusion protein in the present invention is a PEGylated protein that contributes to a desired in vitro or in vivo bioactivity or that is insusceptible to natural actions that would compromise the activity of the fusion protein, such as formation of antibodies, nonspecific adherence to cells or biological surfaces, or degradation or elimination.

A PEG moiety can be attached to the N-terminal amino acid, a cysteine residue (either native or non-native), lysines, or other native or non-native amino acids in a protein's primary sequence. Chemistries for peptide and protein PEGylation have been extensively reviewed (Roberts et al. Adv. Drug Deliv. Rev. 54(4):459-476 (2002)). In addition, specific peptide sequences may be introduced to the primary sequence such that the peptide may be selectively modified by a PEG moiety through a sequence specific enzymatic reaction. Alternatively, a specific peptide sequence may be first modified by a chemically modified group, followed by PEG attachment at the modified group.

Cysteine residues in many proteins may be sequestered in disulfide bonds and are not preferred or available for derivatization. An additional cysteine may be introduced at a location wherein it does not substantially negatively affect the biological activity of the protein, by insertion or substitution through site directed mutagenesis. The free cysteine will serve as the site for the specific attachment of a PEG molecule, thus avoiding the product heterogeneity often observed with amine-specific PEGylation. The preferred site for the added cysteine is exposed on the protein surface and is accessible for PEGylation. The terminal region, C-terminal region, and the linker region of the fusion proteins are potential sites for the cysteine substitution or insertion.

It is also possible to genetically introduce two or more additional cysteines that are not able to form disulfide bonds. In such cases more than one PEG moiety may be specifically attached to the protein. Alternatively, a native, non-essential disulfide bond may be reduced, thus providing two free cysteines for thiol-specific PEGylation.

Free thiol groups may also be introduced by chemical conjugation of a molecule that contains a free cysteine or a thiol group, which may alternatively be modified with a reversible thiol blocking agent.

PEGylation may also be accomplished by using enzyme catalyzed conjugation reactions. One such approach is to use transglutaminases, a family of proteins that catalyze the formation of a covalent bond between a free amine group and the gamma-carboxamide group of protein- or peptide-bound glutamine. Examples of this family of proteins include transglutaminases of many different origins, including thrombin, factor XIII, and tissue transglutaminase from human and animals. A preferred embodiment comprises the use of a microbial transglutaminase, to catalyze a conjugation reaction between a protein substrate containing a glutamine residue embedded within a peptide sequence of LLQG and a PEGylating reagent containing a primary amino group (Sato Adv. Drug Deliv. Rev. 54(4):487-504 (2002)).

Another enzyme-catalyzed PEGylation method involves the use of sortases, a family of enzymes from gram-positive bacteria that can recognize a conserved carboxylic sorting motif and catalyze a transpeptidation reaction to anchor surface proteins to the cell wall envelope (Dramsi et al., Res. Microbiol. 156(3):289-297 (2005)). A preferred embodiment comprises the use of a S. aureus sortase to catalyze a transpeptidation reaction between a protein that is tagged with LPXTG or NPQTN, respectively for sortase A and sortase B, and a PEGylating reagent containing a primary amino group (WO06013202A2). The peptide substrate sequences listed above are for example and non-limiting. It is known in the art that these families of enzymes can recognize and utilize different sequences as substrates, and those sequences are included here as embodiments for the present invention. The preferred peptide substrate sequences listed above are for example and non-limiting. It is known in the art that these families of enzymes can recognize and utilize different sequences as substrates, and those sequences are included here as embodiments for the present invention.

Multifunctional PEGs

While a majority of the PEGylated proteins currently available have one or more PEGs per protein, it is also possible to construct protein conjugates with two or more proteins attached to one PEG moiety. Heterofunctional PEGs are commercially available, and may be used to covalently link two proteins, or any two moieties of a protein.

Preferred PEGylation Sites

Because both toxins and activators possess regions or domains that are important for their respective functions, the attachment of the bulky PEG substituents on these domains may be detrimental to their function. Accordingly a preferred embodiment of the present invention is a PEGylating fusion protein wherein the PEG substituent is situated at a position remote from the catalytic site of an activator (either a protoxin activator or a proactivator activator) and the cell surface target recognition surface of a cell-targeting moiety; and in the case of a protoxin, is not situated within the translocation and catalytic domains of the protoxin, because these domains are expected to be involved in translocation through the plasma membrane and/or to be imported into cytoplasm and PEGylation may prevent such translocations.

In one embodiment of the present invention, the preferred sites of PEGylation are located at or near the N- or C-terminal extremities of proteinaceous cell-targeting moieties. In another embodiment of the present invention, PEGylation is directed to a linker region between different moieties within the fusion protein.

In another embodiment of the present invention, reversible PEGylation may be used.

D. Clearing Agents

The invention optionally also includes the use of clearing agents to facilitate the removal of systemic protease fusion protein prior to the administration of toxin fusion protein. The use of clearing agents in ADEPT therapy is well known in the art (see, for example, Syrigos and Epenetos, Anticancer Res. 19:605 (1999)) and may be utilized in the invention.

IV. Linkages

According to the present invention, each moiety within a protoxin fusion protein (e.g., one or more cell targeting moieties, one or more selectively modifiable activations domains, one or more natively activatable domain, and one or more toxin domains) or a protoxin activator fusion, (e.g., one or more cell targeting moieties, one or more modification domains, one or more natively activatable domain, and one or more toxin domains) may function independently but each is operably linked. Within each fusion protein the operable linkage between the two functional moieties acts as a molecular bridge, which may be covalent or non-covalent. The moieties of each fusion protein may be operably linked in any orientation with respect to each other, that is, C-terminal of one to N-terminal of the other, or C-terminal of one to C-terminal of the other, or N-terminal of one to N-terminal of the other, or by internal residues to terminal residues or internal residues to internal residues. An optional linker can serve as a glue to physically join the two moieties, as a separator to allow spatial independence, or as a means to provide additional functionality to each other, or a combination thereof. For example, it may be desirable to separate the cell-targeting moiety from the operably linked enzyme moiety to prevent them from interfering with each other's activity. In this case the linker provides freedom from steric conflict between the operably linked moieties. The linker may also provide, for example, lability to the connection between the two moieties, an enzyme cleavage site (e.g., a cleavage site for protease or a hydrolytic site for esterase), a stability sequence, a molecular tag, a detectable label, or various combinations thereof.

Chemical activation of amino acid residues can be carried out through a variety of methods well known in the art that result in the joining of the side chain of amino acid residues on one molecule with side chains of residues on another molecule, or through the joining of side chains to the alpha amino group or by the joining of two or more alpha amino groups. Typically the joining induced by chemical activation is accomplished through a linker which may be a small molecule, an optionally substituted branched or linear polymer of identical or nonidentical subunits adapted with specific moieties at two or more termini to attach to polypeptides or substitutions on polypeptides, or an optionally substituted polypeptide. Examples of common covalent protein operable linkage are publically available, including those offered for sale by Pierce Chemical Corporation. In general it is preferable to be able to induce operable linkage of components in a site-specific manner, to afford a simple reproducibly manufactured substance. Operable linkage by chemical activation can be the result of chemical activation targeted to specific residues that are functionally unique i.e. are present only once in the moiety to be activated or are preferentially activatable because of a unique chemical environment, for example, such as would produce a reduction in pK of an epsilon amino unit of a lysine residue. Potential groups for chemical activation can be made functionally unique by genetic removal of all other residues having the same properties, for example to remove all but a single cysteine residue, or all but a single lysine reside. Amino terminal residues can be favorably targeted by virtue of the low pK of the alpha amino group, or by suitable chemistry exploiting the increased reactivity of the alpha amino group in close proximity to another activatable group. Examples of the latter include native chemical ligation, Staudinger ligation, and oxidation of amino terminal serine to afford an aldehyde substituent. Chemical activation can also be carried out through reactions that activate naturally occurring protein substituents, such as oxidation of glycans, or other naturally occurring protein modifications such as those formed by biotin or lipoic acid, or can be based on chemical reactions that convert the functionality of one side chain into that of another, or that introduce a novel chemical reactive group that can subsequently activated to produce the desired operable linkage. Examples of the latter include the use of iminodithiolane to endow a lysine residue with a sulfhydryl moiety or the reaction of a cysteine moiety with an appropriate maleimide or haloacetamide to change the functionality of the thiol to another desired reactive moiety. Chemical activation can also be carried out on both species to be operably linked to provide reactive species that interact with one another to provide an operable linkage, for example the introduction of a hydrazide, hydrazine or hydroxylamine on one moiety and an aldehyde on the other.

Noncovalent operable linkage can be obtained by providing a complementary surface between one moiety and another to provide a complex which is stable for the intended useful persistence of the operably linked moieties in therapeutic use. Such noncovalent linkages can be created from either two or more polypeptides that may be the same or dissimilar or one or more polypeptide and a small molecule or ligand attached to the second moiety. Attachment of the small molecule or ligand can take place through in vitro or in vivo processes, such as the incorporation of biotin or lipoic acid into their specific acceptor sequences which may be natural or artificial biotin or lipoic acid acceptor domains and which may be achieved either by natural incorporation in vivo or by enzymatic biotinylation or lipoylation in vitro. Alternatively, the protein may be substituted with biotin or other moieties by chemical reaction with biotin derivatives. Common examples of biotin derivatives used to couple with proteins include aldehydes, amines, haloacetamides, hydrazides, maleimides, and activated esters, such as N-hydroxysuccinimide esters, Examples of commonly employed noncovalent linkage include the linkage induced by binding of biotin and its derivatives or biotin-related substituents such as iminobiotin or diaminobiotin or thiobiotin to streptavidin or avidin or variants thereof, the binding of enzymes to their covalent or noncovalent specific inhibitors, such as the binding of methotrexate to mammalian dihydrofolate reductase, the binding of natural or synthetic leucine zippers to one another, the binding of enzymes to specific or nonspecific inhibitors, such as antitrypsin or leupeptin or alpha-2-macroglobulin, the binding of aryl bis-arsenates to alpha helices bearing appropriately positioned cysteine residues, the binding between a nucleic acid aptamer and its target; between a peptide and a nucleic acid such as Tat-TAR interaction.

Enzymatic activation of one polypeptide to afford coupling with another polypeptide can also be employed. Enzymes or enzyme domains that undergo covalent modification by reaction with substrate-like molecules can also be used to create fusions. Examples of such enzymes or enzyme domains include O6-alkylguanine DNA-alkyltransferase (Gronemeyer et al. Protein Eng Des Sel. 2006 19(7):309-16), thymidylate synthase, or proteases that are susceptible to covalent or stable noncovalent modification of the active site, as for example DPPIV (SEQ ID NO:65).

The present invention also features the use of bifunctional or multifunctional linkers, which contain at least two interactive or reactive functionalities that are positioned near or at opposite ends, each can bind to or react with one of the moieties to be linked. The two or more functionalities can be the same (i.e., the linker is homobifunctional) or they can be different (i.e., the linker is heterobifunctional). A variety of bifunctional or multifunctional cross-linking agents are known in the art are suitable for use as linkers. For example, cystamine, m-maleimidobenzoyl-N-hydroxysuccinimide-ester, N-succinimidyl-3-(2-pyridyldithio)-propionate, methylmercaptobutyrimidate, dithiobis(2-nitrobenzoic acid), and many others are commercially available, e.g., from Pierce Chemical Co. Rockford, Ill. Additional chemically orthogonal reactions suitable for such specific operable linkage reactions include, for example, Staudinger ligation, Cu[I] catalyzed [2+3] cycloaddition, and native ligation.

The bifunctional or multifunctional linkers may be interactive but non-reactive. Such linkers include the composite use of any examples of non-covalent interactions discussed above.

The length and composition of the linker can be varied considerably provided that it can fulfill its purpose as a molecular bridge. The length and composition of the linker are generally selected taking into consideration the intended function of the linker, and optionally other factors such as ease of synthesis, stability, resistance to certain chemical and/or temperature parameters, and biocompatibility. For example, the linker should not significantly interfere with the regulatory ability of the cell-targeting moiety relating to targeting of the toxin, or with the activity of the toxin or enzyme relating to activation and/or cytotoxicity.

Linkers suitable for use according to the present invention may be branched, unbranched, saturated, or unsaturated hydrocarbon chains, including peptides as noted above.

Furthermore, if the linker is a peptide, the linker can be attached to the toxin moiety and enzyme moiety and/or the cell-targeting moiety using recombinant DNA technology.

In one embodiment of the present invention, the linker is a branched or unbranched, saturated or unsaturated, hydrocarbon chain having from 1 to 100 carbon atoms, wherein one or more of the carbon atoms is optionally replaced by —O— or —NR— (wherein R is H, or C1 to C6 alkyl), and wherein the chain is optionally substituted on carbon with one or more substituents selected from the group of (C1-C6) alkoxy, (C3-C6) cycloalkyl, (C1-C6) alkanoyl, (C1-C6) alkanoyloxy, (C1-C6) alkoxycarbonyl, (C1-C6) alkylthio, amide, azido, cyano, nitro, halo, hydroxy, oxo (═O), carboxy, aryl, aryloxy, heteroaryl, and heteroaryloxy.

Examples of suitable linkers include, but are not limited to, peptides having a chain length of 1 to 100 atoms, and linkers derived from groups such as ethanolamine, ethylene glycol, polyethylene with a chain length of 6 to 100 carbon atoms, polyethylene glycol with 3 to 30 repeating units, phenoxyethanol, propanolamide, butylene glycol, butyleneglycolamide, propyl phenyl, and ethyl, propyl, hexyl, steryl, cetyl, and palmitoyl alkyl chains.

In one embodiment, the linker is a branched or unbranched, saturated or unsaturated, hydrocarbon chain, having from 1 to 50 carbon atoms, wherein one or more of the carbon atoms is optionally replaced by —O— or —NR— (wherein R is as defined above), and wherein the chain is optionally substituted on carbon with one or more substituents selected from the group of (C1-C6) alkoxy, (C1-C6) alkanoyl, (C1-C6) alkanoyloxy, (C1-C6) alkoxycarbonyl, (C1-C6) alkylthio, amide, hydroxy, oxo (═O), carboxy, aryl and aryloxy.

In another embodiment, the linker is an unbranched, saturated hydrocarbon chain having from 1 to 50 carbon atoms, wherein one or more of the carbon atoms is optionally replaced by —O— or —NR— (wherein R is as defined above), and wherein the chain is optionally substituted on carbon with one or more substituents selected from the group of (C1-C6) alkoxy, (C1-C6) alkanoyl, (C1-C6) alkanoyloxy, (C1-C6) alkoxycarbonyl, (C1-C6) alkylthio, amide, hydroxy, oxo (═O), carboxy, aryl and aryloxy.

In a specific embodiment of the present invention, the linker is a peptide having a chain length of 1 to 50 atoms. In another embodiment, the linker is a peptide having a chain length of 1 to 40 atoms.

As known in the art, the attachment of a linker to a protoxin moiety (or of a linker element to cell-targeting moiety or a cell-targeting moiety to a protoxin moiety) need not be a particular mode of attachment or reaction. Various non-covalent interactions or reactions providing a product of suitable stability and biological compatibility are acceptable.

One preferred embodiment of the present invention relies on enzymatic reaction to provide an operable linkage between the moieties of a protoxin, protoxin activator, or protoxin proactivator. Among the enzymatic reactions that produce such operable linkage, it is well-known in the art that transglutaminase ligation, sortase ligation, and intein-mediated ligation provide for high specificity.

The preferred peptide substrate sequences listed above are for example and non-limiting. It is known in the art that these families of enzymes can recognize and utilize different sequences as substrates, and those sequences are included here as embodiments for the present invention.

In some aspects, the invention features the use of natively activatable linkers. Such linkers are cleaved by enzymes of the complement system, urokinase, tissue plasminogen activator, trypsin, plasmin, or another enzyme having proteolytic activity may be used in one embodiment of the present invention. According to another embodiment of the present invention, a protoxin is attached via a linker susceptible to cleavage by enzymes having a proteolytic activity such as a urokinase, a tissue plasminogen activator, plasmin, thrombin or trypsin. In addition, protoxins may be attached via disulfide bonds (for example, the disulfide bonds on a cystine molecule) to the cell-targeting moiety. Since many tumors naturally release high levels of glutathione (a reducing agent) this can reduce the disulfide bonds with subsequent release of the protoxin at the site of delivery.

In one embodiment, the cell-targeting moiety is linked to a protoxin by a cleavable linker region. In another embodiment of the invention, the cleavable linker region is a protease-cleavable linker, although other linkers, cleavable for example by small molecules, may be used. Examples of protease cleavage sites are those cleaved by factor Xa, thrombin and collagenase. In one embodiment of the invention, the protease cleavage site is one that is cleaved by a protease that is up-regulated or associated with cancers in general. Examples of such proteases are uPA, the matrix metalloproteinase (MMP) family, the caspases, elastase, and the plasminogen activator family, as well as fibroblast activation protein. In still another embodiment, the cleavage site is cleaved by a protease secreted by cancer-associated cells. Examples of these proteases include matrix metalloproteases, elastase, plasmin, thrombin, and uPA. In another embodiment, the protease cleavage site is one that is up-regulated or associated with a specific cancer. In yet another embodiment, the proteolytic activity may be provided by a protease fusion targeted to the same cell. Various cleavage sites recognized by proteases are known in the art and the skilled person will have no difficulty in selecting a suitable cleavage site. Non-limiting examples of cleavage sites are provided elsewhere in this document. As is known in the art, other protease cleavage sites recognized by these proteases can also be used. In one embodiment, the cleavable linker region is one which is targeted by endocellular proteases.

Chemical linkers may also be designed to be substrates for carboxylesterases, so that they may be selectively cleaved by these carboxyltransferases or corresponding fusion proteins with a cell-targeting moiety. One preferred embodiment comprises the use of a carboxyl transferase activity to activate the cleavage of an ester linker. For example but without limitation, secreted human carboxyltransferase-1, -2, and -3 may be used for this purpose. Additional examples include carboxyl transferase of other origins.

Another embodiment of the cleavable linkers comprises nucleic acid units that are specifically susceptible to endonucleases. Endonucleases are known to be present in human plasma at high levels.

In another embodiment, the modifiable activation moiety is not a peptide, but a cleavable linker that may be acted upon by a cognate enzymatic activity provided by the activator or proactivator. The cleavable linker is preferably situated at the same location as the furin-like cleavage sequence in an activatable protoxin, or at the location of the zymogen inhibitory peptide in an activatable proactivator. The cleavable linker may replace the furin-like cleavage sequence or be attached in parallel to the furin-like cleavage or another modifiable activation moiety, providing a protoxin that requires both a furin-like cleavage or other proteolytic event and a linker cleavage for activation. In one embodiment the cleavable linker joins the ADP ribosyltransferase domain of a DT-based protoxin to the translocation domain of that or another protoxin. In another embodiment the cleavable linker joins the translocation domain of a PEA or VCE-based protoxin to the ADP ribosyltransferase domain of the same or a different toxin. In yet another embodiment the cleavable linker joins the pore-forming domain of a pore-forming toxin with the C-terminal inhibitory peptide.

Preferable cleavable linkers are those which are stable to in vivo conditions but susceptible to the action of an activator. Many examples of suitable linkers have been provided in the context of attempts to develop antibody-directed enzyme prodrug therapy. For example a large class of enzyme substrates that lead to release of an active moiety, such as a fluorophore, have been devised through the use of what are known as self-immolative linkers. Self-immolative linkers are designed to liberate an active moiety upon release of an upstream conjugation linkage, for example between a sugar and an aryl moiety. Such linkers are often based on glycosides of aryl methyl ethers, for example the phenolic glycosides of 3-nitro, 4-hydroxy benzyl alcohol; see for example Ho et al. Chembiochem, Mar. 26, 2007; 8(5):560-6, or the phenolic amides of 4-amino benzyl alcohol, for example Niculescu-Duvaz et al. J Med Chem. Dec. 17, 1998; 41(26):5297-309 or Toki et al. J Org Chem. Mar. 22, 2002; 67(6):1866-72.

To create self-immolative linkers based on glycosides the phenolic hydroxyl is glycated by reaction with a 1-Br-substituted sugar such as alpha-1-Br galactose or alpha-1-Br glucuronic acid to provide the substrate for the activating enzyme, and the benzyl alcohol moiety is then activated with a carbonylation reagent such as phosgene or carbonyl diimidazole and reacted with a primary amine to afford a carbamate linkage. Upon scission of the aryl glycosidic bond or the aryl ester, the aryl moiety eliminates, leaving a carbamoyl moiety that in turn eliminates, affording CO2 and the regenerated amine. Said amine may be the alpha amino group of a polypeptide chain or the epsilon amino of a lysine side chain.

To create self-immolative linkers based on amide bonds the phenyl amine of 4-amino benzyl alcohol is reacted with an activated carboxyl group of a suitable peptide or amino acid to create a phenyl amide that can be a substrate for an appropriate peptidase, for example carboxypeptidase G2 Niculescu-Duvaz et al. J Med Chem. 41(26):5297-309 (1998). The benzyl alcohol moiety is then activated with a carbonylation reagent such as phosgene or carbonyl diimidazole and reacted with a primary amine to afford a carbamate linkage. Upon scission of the aryl amide bond, the aryl moiety eliminates, leaving a carbamoyl moiety that in turn eliminates, affording CO2 and the regenerated amine. Said amine may be the alpha amino group of a polypeptide chain or the epsilon amino of a lysine side chain.

For the creation of an appropriate self-immolating activation moiety according to the present invention the aryl group is substituted with a reactive moiety that provides a linkage to one element of the protoxin or proactivator, such as the toxin moiety or the translocation moiety or the inhibitory peptide moiety.

Similar forms of self-immolative linker are also well-known in the art. For example Papot et al. Bioorg Med Chem Lett. 8(18):2545-8 (1998) teach the creation of glucuronide prodrugs based on aryl malonaldehydes that undergo elimination of the aryl linker moiety upon cleavage by a glucuronidase. Suitable linkers based on aryl malonaldehydes in the context of the present invention provide a modifiable activation moiety in which the aryl substituent is operably linked to one terminus of the toxin moiety, for example at the location of the furin cleavage site, and the carbamoyl functionality is operably linked to the translocation moiety or inhibitory moiety. In the system devised by Papot et al, cleavage by glucuronidase will result in elimination of the aryl malonaldehyde and activation of the protoxin. Similar elimination events are known to take place following hydrolysis of the lactam moiety of linkers based on 7-aminocephalosporanic acid, and enzymatically activated prodrugs based on beta-lactam antibiotics or related structures are well known in the art. For example Alderson et al. Bioconjug Chem. 17(2):410-8 (2006) teach the creation of a 7-aminocephalosporanic acid-based linker that undergoes elimination and scission of a carbamate moiety in similar fashion to that of the aryl malonaldehydes disclosed by Papot et at. In addition, Harding et al. Mol Cancer Ther. 4(11): 1791-800 (2005) teach a beta-lactamase that has reduced immunogenicity that can be favorably applied as an activator for a prodrug moiety based on a 7-aminocephalosporanic acid nucleus.

In yet another embodiment the modifiable activation moiety is a peptide but is operably linked by a flexible nonpeptide linker at either or both termini in the same location as the natural furin-like protease cleavage site, or in parallel to the natural furin-like cleavage site. In such embodiments the activator is a cognate protease or peptide hydrolase recognizing the peptide of the modifiable activation moiety. In a doubly triggered protoxin, the furin-like cleavage site is replaced by a modifiable activation moiety and a cleavable linker is attached in parallel to the modifiable activation moiety. In such a protoxin the action of two activators is required to activate the protoxin.

V. Isolation and Purification of Toxin Fusion and Protease Fusion Proteins

A. General Strategies for Recombinant Protein Purification

There are many established strategies to isolate and purify recombinant proteins known to those skilled in the art, such as those described in Current Protocols in Protein Science (Coligan et al., eds. 2006). Conventional chromatography such as ion exchange chromatography, hydrophobic-interaction (reversed phase) chromatography, and size-exclusion (gel filtration) chromatography, which exploit differences of physicochemical properties between the desired recombinant protein and contaminants, are widely used. HPLC can also been used.

To facilitate the purification of recombinant proteins, a variety of vector systems have been developed to express the target protein as part of a fusion protein appended by an N-terminal or C-terminal polypeptide (tag) that can be subsequently removed using a specific protease. Using such tags, affinity chromatography can be applied to purify the proteins. Examples of such tags include proteins and peptides for which there is a specific antibody (e.g., FLAG fusion purified using anti-FLAG antibody columns), proteins that can specifically bind to columns containing a specific ligand (e.g., GST fusion purified by glutathione affinity gel), polyhistidine tags with affinity to immobilized metal columns (e.g., 6 His tag immobilized on Ni²⁺ column and eluted by imidazole), and sequences that can be biotinylated by the host during expression or in vitro after isolation and enable purification on an avidin column (e.g., BirA).

B. Isolation and Purification of Fusion Proteins Expressed in Insoluble Form

Many recombinant fusion proteins are expressed as inclusion bodies in Escherichia coli, i.e., dense aggregates that consist mainly of a desired recombinant product in a nonnative state. In fact, most reported DT-ScFv fusion proteins expressed in E. coli are obtained in insoluble forms. Usually the inclusion bodies form because (a) the target protein is insoluble at the concentrations being produced, (b) the target protein is incapable of folding correctly in the bacterial environment, or (c) the target protein is unable to form correct disulfide bonds in the reducing intracellular environment.

Those skilled in the art recognize that different methods that can be used to obtain soluble, active fusion proteins from inclusion bodies. For example, inclusion bodies can be separated by differential centrifugation from other cellular constituents to afford almost pure insoluble product located in the pellet fraction. Inclusion bodies can be partially purified by extracting with a mixture of detergent and denaturant, either urea or guanidine.HCl, followed by gel filtration, ion exchange chromatography, or metal chelate chromatography as an initial purification step in the presence of denaturants. The solubilized and partially purified proteins can be refolded by controlled removal of the denaturant under conditions that minimize aggregation and allow correct formation of disulfide bonds. To minimize nonproductive aggregation, low protein concentrations should be used during refolding. In addition, various additives such as nondenaturing Concentrations of urea or guanidine.HCl, arginine, detergents, and PEG can be used to minimize intermolecular associations between hydrophobic surfaces present in folding intermediates.

C. Isolation and Purification of Fusion Proteins Expressed in Soluble Form

Recombinant proteins can also be expressed and purified in soluble form. Recombinant proteins that are not expressed in inclusion bodies either will be soluble inside the cell or, if using an excretion vector, will be extracellular (or, if E. coli is the host, possibly periplasmic). Soluble proteins can be purified using conventional methods afore described.

VI. Assays for Measuring Inhibition of Cell Growth

Various assays well known in the art are useful for determining the efficacy of the protein preparations of the invention, including those assays that measure cell proliferation and death. For example, it has been shown that one molecule of diphtheria toxin catalytic fragment (DTA) introduced into the cytosol of a cell is sufficient to prevent the cell from multiplying and forming a colony (Yamaizumi et al., Cell 15:245 (1978)). The following are examples of many assays that can be used, alone or in combination, for analyzing the cytotoxicity of the reagents in the present invention.

A. Protein Synthesis Inhibition Assays

Because many toxins (e.g., DT) exert their cytotoxicity through inhibition of protein synthesis, an assay that directly quantifies protein being synthesized by the cell after its exposure to the toxin is especially useful. In this assay, cells are exposed to a toxin and then incubated transiently with radioactive amino acids such as [³H]-Leu, [³⁵S]-Met or [³⁵S]-Met-Cys. The amount of radioactive amino acid incorporated into protein is subsequently determined, usually by lysing cells and precipitating proteins with 10% trichloroacetic acid (TCA), providing a direct measure of how much protein is synthesized. Using such an assay, it was demonstrated that, although the entry of DT into a cell is not associated with an immediate block in protein synthesis, prolonged action (4-24 hours) of single DT catalytic fragment molecules in the cytosol is sufficient to obtain complete protein synthesis inhibition at low toxin concentrations (Falnes et al., J. Biol. Chem. 275:4363 (2000)).

An extension of this method is a luciferase-based assay (Zhao and Haslam, J. Med. Microbiol. 54:1023 (2005)). Luciferase cDNA was incorporated into a wide variety of dividing or non-dividing mammalian cells using an adenoviral expression system, and the resulting cells allowed to constitutively transcribe the luciferase cDNA, which had been engineered to contain an additional PEST sequence for a short intracellular half-life. The assay measures the level of protein synthesis in cells through the light output from D-luciferin reaction catalyzed by the short-lived luciferase. In cells constitutively expressing the luciferase mRNA, inhibition of protein synthesis results in diminished luciferase translation and proportionately reduced light output.

B. Thymidine Incorporation Assay

The rate of proliferation of cells can be measured by determining the incorporation of [³H]-thymidine into cellular nucleic acids. This assay may be used for analyzing cytotoxicity of toxins (e.g., DT-based immunotoxins). Using this method a DT-IL3 immunotoxin was shown to be active in inhibiting growth of IL3-receptor bearing human myeloid leukemia cell lines (Frankel et al., Leukemia. 14:576 (2000)). The toxin fusion and protease fusion proteins of the present invention may be tested using such an assay, individually or combinatorially.

C. Colony Formation Assay

Colony formation may provide a much more sensitive measure of toxicity than certain other commonly employed methods. The reason for this increased sensitivity may be the fact that colony formation is assessed while the cells are in a state of proliferation, and thus more susceptible to toxic effects. The sensitivity of the colony-formation assay, and the fact that dose and time-dependent effects are detectable, enables acute and chronic exposure periods to be investigated as well as permitting recovery studies. For example, the cytotoxicity of a recombinant DT-IL6 fusion protein towards human myeloma cell lines was investigated using methylcellulose colony formation by U266 myeloma cells. In cultures containing both normal bone marrow and U266 cells DT-IL-6 effectively inhibited the growth of U266 myeloma colonies but had little effect on normal bone marrow erythroid, granulocyte and mixed erythroid/granulocyte colony growth (Chadwick et al., Haematol. 85:25 (1993)).

D. MTT Cytotoxicity Assay

The cytotoxicity of a particular fusion protein or a combination of fusion proteins can be assessed using an MTT cytotoxicity assay. The specific cytotoxicity of a DT-GMCSF fusion protein against human leukemia cell lines bearing high affinity receptors for human GMCSF was demonstrated using such an MTT assay, colony formation assay, and protein inhibition assay (Bendel et al., Leuk. Lymphoma. 25:257 (1997)). In a typical MTT assay, the yellow tetrazolium salt (MTT) is reduced in metabolically active cells to form insoluble purple formazan crystals, which are solubilized by the addition of a detergent and quantified by UV-VIS spectrometry. After cells are grown to 80-100% confluence, they are washed with serum-free buffer and treated with cytotoxic agent(s). After incubation of the cells with the MTT reagent for approximately 2 to 4 hours, a detergent solution is added to lyse the cells and solubilize the colored crystals. The samples are analyzed at a wavelength of 570 nm and the amount of color produced is directly proportional to the number of viable cells.

VII. Functional Assays for DT and Protease Fusion Proteins

A. In Vitro Protein Synthesis Inhibition Assay

In eukaryotic cells, DT inhibits protein synthesis because its catalytic domain can inactivate elongation factor 2 (EF-2) by catalyzing its ADP-ribosylation after endocytosis to cytosol. In vitro eukaryotic translation systems, e.g., using rabbit reticulocyte lysate and wheat germ extract, are potentially suited for examining the catalytic function of recombinant DT fusion proteins. For example, TNT-coupled wheat germ extract, supplemented by NAD⁺, amino acids, [³⁵S]-Met, DNA template, and an RNA polymerase, is used to test the inhibition of protein synthesis by a recombinantly expressed catalytic fragment of DT (Epinat and Gilmore, Biochim. Biophys. Acta. 1472:34 (1999)). The level of S-labeled translated protein is an indicator of the extent of DT toxicity.

Because in vitro inhibition of protein synthesis does not require endocytosis of full length DT, it has been shown that its proteolytic activation increased ADP-ribosylation of EF-2 (Drazin et al., J. Biol. Chem. 246:1504 (1971)). Thus these in vitro assays can be used to screen inhibitory effects of DT fusions in the absence or presence of certain proteolytic activity, providing a facile assay to analyze the functional integrity of engineered DT fusion proteins as well as that of protease fusion proteins.

B. In Vitro EF-2 ADP-Ribosylation Assay

DT inhibits protein synthesis by catalyzing the transfer of ADP-ribose moiety of NAD to a post-translationally modified His715 of EF-2 called diphthamide. Thus the function of DT fusions can also be directly assayed in vitro by correlating its catalytic activity to rate of transfer of radiolabeled ADP-ribose to recombinant EF-2 (Parikh and Schramm, Biochemistry 43:1204 (2004)). This assay has been applied for testing the inhibition of ADP-ribosyltransferase activity, and is often used as one of the assays for DT-based immunotoxins (Frankel et al., Leukemia. 14:576 (2000)). Non-radioactively labeled NAD, such as biotinylated NAD or etheno-NAD, may also be used as a substrate (Zhang. Method Enzymol. 280:255-265 (1997)).

C. In Vitro Proteolytic Activity Assay

The functional activity of recombinant protease fusion proteins may be assayed in vitro either using a peptide or protein substrate containing the recognition sequence of the protease. Various protocols are well known to those skilled in the art.

VIII. Administration of Fusion Proteins

The fusion proteins of the invention are typically administered to the subject by means of injection using any route of administration such as by intrathecal, subcutaneous, submucosal, or intracavitary injection as well as by intravenous or intraarterial injection. Thus, the fusion proteins may be injected systemically, for example, by the intravenous injection of the fusion proteins into the patient's bloodstream or alternatively, the fusion proteins can be directly injected at a specific site.

The protoxin of the invention can be administered prior to, simultaneously with, or following the administration of the protoxin activator or protoxin proactivator and optionally administered prior to, simultaneously with, or following the administration of the proactivator activator of the invention. In preferred embodiments the components are administered in such a way as to minimize spontaneous activation during administration. When administered separately, the administration of two or more fusion proteins can be separated from one another by, for example, one minute, 15 minutes, 30 minutes, one hour, two hours, six hours, 12 hours, one day, two days, one week, or longer. Furthermore, one or more of the fusion proteins of the invention may be administered to the subject in a single dose or in multiple doses. When multiple doses are administered, the doses may be separated from one another by, for example, one day, two days, one week, two weeks, or one month. For example, the fusion proteins may be administered once a week for, e.g., 2, 3, 4, 5, 6, 7, 8, 10, 15, 20, or more weeks. It is to be understood that, for any particular subject, specific dosage regimes should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the fusion proteins. For example, the dosage of the fusion proteins can be increased if the lower dose does not sufficiently destroy or inhibit the growth of the desired target cells. Conversely, the dosage of the fusion proteins can be decreased if the target cells are effectively destroyed or inhibited.

While the attending physician ultimately will decide the appropriate amount and dosage regimen, a therapeutically effective amount of the fusion proteins may be, for example, in the range of about 0.0035 μg to 20 μg/kg body weight/day or 0.010 μg to 140 μg/kg body weight/week. A therapeutically effective amount may be in the range of about 0.025 μg to 10 μg/kg, for example, about 0.025, 0.035, 0.05, 0.075, 0.1, 0.25, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 5.0, 6.0, 7.0, 8.0, or 9.0 μg/kg body weight administered daily, every other day, or twice a week. In addition, a therapeutically effective amount may be in the range of about 0.05, 0.7, 0.15, 0.2, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 10.0, 12.0, 14.0, 16.0, or 18.0 μg/kg body weight administered weekly, every other week, or once a month. Furthermore, a therapeutically effective amount of the fusion proteins may be, for example in the range of about 100 μg/m² to 100,000 μg/m² administered every other day, once weekly, or every other week. The therapeutically effective amount may be in the range of about 1000 μg/m² to 20,000 μg/m², for example, about 1000, 1500, 4000, or 14,000 μg/m² of the fusion proteins administered daily, every other day, twice weekly, weekly, or every other week.

In some cases it may be desirable to modify the plasma half-life of a component of the combinatorial therapeutic agent of the present invention. The plasma half-lives of therapeutic proteins have been extended using a variety of techniques such as those described by Collen et al., Bollod 71:216-219 (1998); Hotchkiss et al., Thromb. Haemostas. 60:255-261 (1988); Browne wt al., J. Biol. Chem. 263:1599-1602 (1988); Abuchowski et al., Cancer Biochem. Biophys. 7:175 (1984)). Antibodies have been chemically conjugated to toxins to generate immunotoxins which have increased half-lives in serum as compared with unconjugated toxins and the increased half-life is attributed to the native antibody. WO94/04689 teaches the use of modified immunotoxins in which the immunotoxin is linked to IgG constant region domain having the property of increasing the half-life of the protein in mammalian serum. The IgG constant region domain is CH2 or a fragment thereof.

The administration the fusion proteins of the invention may be by any suitable means that results in a concentration of the fusion proteins that, combined with other components, effectively destroys or inhibits the growth of target cells. The fusion proteins may be contained in any appropriate amount in any suitable carrier substance, and is generally present in an amount of 1-95% by weight of the total weight of the composition. The composition may be provided in a dosage form that is suitable for any parenteral (e.g., subcutaneous, intravenous, intramuscular, topical, or intraperitoneal) administration route. The pharmaceutical compositions are formulated according to conventional pharmaceutical practice (see, e.g., Remington: The Science and Practice of Pharmacy (20th ed.), ed. Gennaro, Williams & Wilkins, 2000 and Encyclopedia of Pharmaceutical Technology, eds. Swarbrick and Boylan, 1988-1999, Marcel Dekker, New York).

IX: Experimental Results

A. Construction of Fusion Proteins and Cell Lines

Construction of a Human Granzyme B-Anti-CD19 ScFv (GrB-Anti-CD19) Fusion Gene

The sequence corresponding to the mature human Granzyme B (amino acids 21 to 247) was amplified from a full length Granzyme B cDNA clone obtained from OriGene Inc. and inserted into the pEAK15 vector together with synthetic anti-CD19 ScFv DNA fragment by a three-piece ligation (pEAK15 GrB-anti-CD19L). The promoter for the fusion gene is a CMV/chicken β-actin hybrid promoter. The open reading frame encoding the fusion protein directs the formation of a signal peptide derived from the Gaussia princeps luciferase, a synthetic N-linked glycosylation site, a FLAG tag and an enterokinase cleavage sequence followed by the mature human granzyme B sequence, a flexible linker (Gly-Gly-Gly-Ser)₃, the anti-CD19 ScFv, and a C-terminal 6 His tag (See FIG. 1A for schematic depiction of the fusion protein). The DNA sequences encoding all fusion proteins were confirmed by DNA sequencing.

Construction of Diphtheria Toxin Anti-CD5 ScFv (DT-Anti-CD5) Fusion Gene

The DT-anti-CD5 fusion gene was made synthetically by Retrogen Co. (San Diego) with codons optimized for expression in Pichia Pastoris and human cell lines. The sequence encoding the furin recognition site (₁₉₀ RVRRSVG₁₉₆ (SEQ ID NO:66)) was replaced with a consensus granzyme B recognition sequence (₁₉₀ IEPDSG₁₉₅ (SEQ ID NO:13)). Two potential N-glycosylation sites were mutated as described (Thompson et al. Protein Eng. 14(12):1035-41 (2001)) and a 6 His tag sequence was added to the C-terminus of the fusion gene for detection and purification. The fusion gene was cloned into XhoI and NotI sites of the pPIC9 vector (Invitrogen) while maintaining the α-factor signal peptide and the Kex2 cleavage site.

Generation of CD19⁺Jurkat, CD5⁺Raji, and CD5⁺JVM3 Cells

Jurkat SVT35 cells were maintained in IMDM (Invitrogen) supplemented with 10% fetal calf serum (Hyclone). JVM-3 (DSMZ, Germany) was maintained in RPMI 1640 (Invitrogen) supplemented with 10% Fetal bovine serum (Hyclone), 2 mM L-Glutamine.

To prepare the recombinant viruses, we replaced the GFP gene in the retroviral vector M3P-GFP with CD19 or CD5 full length cDNA. To produce viral particles, linearized M3P-CD19 plasmid was cotransfected with pMD-MLV, and pMD-VSVG to 293 ETN cells, which were seeded at 5×10⁶ per 10 cm² plate a day before transfection. The DNA concentrations of M3P-CD19, pMD-MLV-G/P and pMD-VSVG were 10 μg, 7 μg and 3 μg, respectively. The volume (μl) of TransFectin was 2.5 times of the total DNA concentration (μg). Viral particles were collected 48 hours after transfection and filtered through a 0.45 μm filter (Corning).

For infection, 5 10⁵ Jurkat cells were suspended in 1.5 ml culture medium and mixed with 1.5 ml filtered virus in a 6-well plate. Three μl of 8 mg/ml polybrene was added to the mixture to the final concentration of 8 μg/ml. The plate was centrifuged at 2000 rpm for 1 hour before culturing in 37° C. incubator containing 5% CO₂. To isolate Jurkat cells expressing CD19, the infected cells were sorted after staining with FITC conjugated anti-human CD19 antibody (Pharmingen, San Diego, Calif. Jurkat cells expressing high concentrations of CD19 were collected and used for the cytotoxicity assay.

Flow Cytometric Analysis

The presence of CD5 and CD19 on cell surface was analyzed using indirect immunofluorescence staining. Cells were first incubated with mouse anti-human CD5 or mouse anti-human CD19 (eBioscience) at a concentration of 0.5 μg per one million cells. Goat F (ab′)₂ anti-mouse IgG1 conjugated with RPEA (Southern Biotechnology) was used as secondary antibody at a concentration of 0.25 μg per million of cells. The stained cells were analyzed by flow cytometry (FAXCaliber).

B. Expression and Purification GrB-Anti-CD19 Fusion from 293ETN Cells

293ETN cells were seeded at 5 10⁶-6 10⁶ cells per 10 cm plate and were transfected with 12 μg of pEAK15 GrB-anti-CD19L and 25 μl of TransFectin (Bio-Rad) according to the manufacturer's protocol. Transfected cells were cultured in Opti-MEM (Invitrogen) for 3 days to allow fusion proteins to accumulate. Supernatants were collected and incubated with pre-equilibrated Ni-NTA resin (Qiagen) and the fusion proteins were eluted with the buffer containing 50 mM HEPES pH7.5, 150 mM NaCl, 250 mM imidazole and 5% glycerol. The purified GrB-anti-CD19 fusion proteins were incubated with enterokinase (New England Biolabs) at room temperature overnight to activate the proteolytic activity of Granzyme B. To remove enterokinase and N-terminal peptide released by enterokinase, the reaction mixture was subjected to affinity purification with Ni-NTA resin. In another form of preparation, the enterokinase and N-terminal peptide released by enterokinase, were removed by gel filtration purification (superdex 200, G E Healthcare). The proteolytic activity of the granzyme B-anti-CD19 ScFv was measured by incubating the purified proteins with a fluorogenic peptide substrate (Ac-IEPD-AMC, Sigma Aldrich). Accumulation of fluorescent product was monitored every 30 s at excitation and emission wavelengths of 380 and 460 nm respectively for 15 min.

C. Expression and Purification of DT-Anti-CD5 Fusion from P. Pastoris

Pichia Pastoris KM71 cells (Invitrogen) were transformed with the expression plasmid by electroporation. Positive clones were selected according to manufacturer's protocol. For large scale purification, a single colony was cultured at 28° C. overnight in 10 ml Buffer Minimal Glycerol pH 6.0 medium (BMG). The overnight culture was transferred to 1 L BMG pH 6.0 and cultured at 28° C. until OD600 reached 6.0. To induce protein expression, the culture was spun down and resuspended with 100 ml Buffered (pH6.6) Methanol-complex Medium containing 1% casamino acids (BMMYC) and cultured at 15° C. for 48 hours. Supernatants were collected and adjusted to pH 7.6 with 5% NaOH. Clarified supernatants were subjected to affinity purification as described above for the purification of the GrB-anti-CD19 fusion protein.

D. Expression and Purification of DT-Anti-CD5, Anti-CD5-PEA, and Anti-CD5-VCE Fusion Proteins from E. Coli

DNA sequence corresponding to αCD5-PEA, αCD5-VCE and their variants were cloned into NcoI and NotI of the pET28 vector (Novagen). Transformed bacterial cells (BL21) were cultured with LB medium at 37° C. To induce expression of insoluble fusion proteins, protein expression was induced with 1 mM IPTG at 37° C. for 4 hours at OD₆₀₀=0.8-1.0. The 40 ml of harvested cell pellet was re-suspended in 5 ml of B-PER II (Pierce) and the inclusion body was purified with B-PER II according the manufacturer's instruction. Purified inclusion body was dissolved with 20 mM Tris 8.0, 150 mM NaCl, 6 M GuCl and 1 mM β-ME and further purified with Ni-NTA resin. Final purified fusion proteins were refolded at the concentration of 0.2 mg/ml with the protocol described previously (Umetsu M. et al. J. Biol. Chem. 278:8979-8987 (2003)). To induce expression of soluble ScFv-VCE fusion proteins, the synthetic genes were cloned into NcoI and NotI of the pET22b vector. Protein expression was induced with 0.2 mM IPTG for overnight at 17° C. at OD60=0.3-0.5. Periplasmic fraction of bacteria was collected as described (Malik et al. Prot. Exp. Pur. Advanced electronic publication (2007)) and fusion protein was purified with Ni-NTA resin.

E. Specific Proteolytic Activity of GrB-Anti-CD19 Fusion Protein

To evaluate the enzymatic activity of purified GrB-anti-CD19 fusion protein, a fluorogenic peptide substrate (Ac-IEPD-AMC) (SEQ ID NO:9) was used to compare the activity of the fusion protein with that of purified mouse granzyme B purchased from Sigma. Purified GrB-anti-CD19 exhibited activity similar to that of the commercial mouse granzyme B preparation, suggesting that addition of a ScFv moiety to the C-terminal of human granzyme B did not impair the proteolytic activity and that enterokinase treatment effectively removed the terminal sequence preceding the first isoleucine of mature granzyme B, allowing the enzymatic activity of the fusion protein to be expressed.

To establish whether the DT-anti-CD5 fusion protein bearing a granzyme B cleavage site could be recognized as a substrate by either mouse granzyme B or GrB-anti-CD19 fusion protein, the DT-anti-CD5 fusion protein containing an N-terminal FLAG tag was incubated with either mouse granzyme B (FIGS. 1B and C, lanes 2) or GrB-anti-CD19 fusion protein (FIG. 1B, lane3). The reaction yielded an N-terminal 25 kD fragment corresponding to the A chain of the diphtheria toxin (FIG. 1B) and a C-terminal 50 kD fragment corresponding the B chain of diphtheria toxin and the ScFv moiety (FIG. 1C), consistent with the interpretation that the DT-anti-CD5 fusion protein could be cleaved specifically at the engineered granzyme B site IEPD↓SG (SEQ ID NO:13).

To further study the cleavage specificity of various DT-anti-CD5 fusion proteins by different proteases, the furin cleavage site of the DT-anti-CD5 fusion protein was replaced with that of a human rhinovirus 3C protease (HRV 3C) cleavage site (ALFQ↓GPLQ) (SEQ ID NO:14) (FIG. 1C, lanes 5 to 8). DT-anti-CD5 bearing an HRV 3C protease cleavage sequence can only be cleaved by HRV 3C protease, not granzyme B or furin (FIG. 1C, lanes 6, 7 and 8). Furthermore, when the furin cleavage site was replaced by a granzyme M recognition site KVPL↓SG SEQ ID NO:67), the resulting toxin DT_(GrM)-anti-CD19 showed synergistic toxicity with fusion protein GrM-anti-CD5 to CD19⁺Jurkat cells (FIG. 14). The toxicity of DT_(GrM)-anti-CD19 suggests that this particular toxin fusion may be more susceptible to activation by endogenous proteolytic activities.

The present results demonstrate that replacing the furin cleavage sequence with other protease cleavage sequences renders the mutant DT inactive (or less active in the case of GrM) and that the mutant DT fusion proteins can be selectively activated by proteases that recognize engineered cleavage sequences.

F. Mutant form of Granzyme B with Altered Cleavage Site Specificity

The redirection of the proteolytic specificity of a protease through mutational alteration of residues surrounding the catalytic pocket is well-known in the art. In particular, previous studies involving the site directed mutagenesis of granzyme B, as well as studies of granzyme B proteins from different species, have identified residues that define the substrate specificity of the enzyme, and have provided mutant forms that have altered cleavage specificity (Harris et al. J. Biol. Chem. 273: 27364-27373 (1998); Ruggles et al. J. Biol. Chem. 279:30751-30759 (2004); Casciola-Rosen et al. J biol. Chem. 282:4545-4552(2007)). Similarly, mouse granzyme B isoforms have been found to exhibit much reduced cleavage activity on human Bid, mouse Bid and human caspase 3 than human granzyme B. As a result, mouse granzyme B is thought to be less likely to induce apoptosis in human cells (Casciola-Rosen et al. J Biol. Chem. 282:4545-4552(2007)). Several mutant forms of granzyme B from the Harris et al. study were presumed to have impaired ability to initiate apoptotic pathway due to their altered cleavage sequence specificity. We generated a fusion protein from one such mutant form of granzyme B in which Asn218 of is replaced with Thr (N218T) and showed that the N218T granzyme B exhibited an cleavage site preference toward IAPD (SEQ ID NO:48), a sequence which is not considered a preferred substrate for the wild type granzyme B. Furthermore, we found that the cleavage activity of N218T toward the IAPD (SEQ ID NO:48) sequence is higher than the cleavage activity of wild type granzyme B toward IEDP (SEQ ID NO:9). Thus, in one embodiment of the present invention, a granzyme B fusion protein can be modified to lessen/abrogate the ability to induce apoptosis of target cells, while possessing full (or improved) proteolytic activity toward the optimal cleavage sequences.

We compared the ability of granzyme B fusion proteins bearing wild type human granzyme B sequence with one bearing the N218T mutation to cleave substrates bearing IEPD (SEQ ID NO:9) or IAPD sequence (SEQ ID NO:48). Under the conditions where only 20% of the substrate was cleaved, we found that N218T cleaved IEPD (SEQ ID NO:9) substrate at comparable capacity as its wild type counterpart (FIG. 28 compare lanes 5 and 6). As expected, we found that N218T cleaved IAPD (SEQ ID NO:48) substrate more efficiently than its wild type counterpart (FIG. 28 compare lanes 5 and 6). Consistent with the in vitro cleavage results, we found that combination of IADP (SEQ ID NO:48) bearing protoxin and N218T mutant granzyme B protoxin activator exhibited higher toxicity to target cells among all the possible combinations of the IEDP/IAPD (SEQ ID NO:48) bearing protoxin and two different forms of granzyme B protoxins activators (data not shown).

G. Cytotoxicity Assay of DT, PEA, or VCE Based Toxin Fusions

The cytotoxicity of combinatorial immunotoxins was tested on cell lines that express both CD5 and CD19, as well as on the corresponding parental cell lines. Cells were placed in a 96-well plate at 5 10⁴ cells per well in 90 μl leucine-free RPMI and were incubated with 10 μl leucine-free RPMI containing various concentrations of GrB-anti-CD19 ScFv and/or DT-anti-CD5 ScFv fusion proteins at 37° C. for 20 hours in 5% CO₂. Inhibition of protein synthesis was measured by adding 0.33 μCi of [³H]-leucine for 1 hour at 37° C. Cells were harvested by filtration onto glass fiber papers by cell harvester (InoTek 96 well cell harvester) and the rate of [³H]-leucine incorporation was determined by scintillation counting. Cell viability was normalized to control wells treated with protein storage buffer. The [³H] incorporation background was obtained by treating cells with 1 mM cycloheximide for 30 min before adding [³H]-leucine. Each point shown represents the average value of duplicate wells.

Combination of GrB-Anti-CD19 and DT-Anti-CD5Fusion Proteins Exhibits Specific Cytotoxicity

Having established the protease fusion protein is functional in vitro, we then asked if the pair of fusion proteins could specifically target cells that express both CD5 and CD19. To this end, we generated a reporter cell B cell line, CD5⁺Raji, expressing CD5 from a human Raji B cell line. Cytometric analyses using anti-CD5 and anti-CD19 antibodies indicated that both CD5 and CD19 were expressed from the CD5⁺Raji cell line (FIG. 2), whereas the parental Raji cells express only CD19. The expression of CD5 from the CD5⁺Raji cell line appeared to be stable, as no significant changes in CD5 level were observed over a long period of culturing.

To evaluate the ability of the fusion proteins to kill specific target cells, we incubated the fusion proteins singly or jointly with either Raji or CD5⁺Raji cells, and then measured protein synthesis activity. We found that GrB-anti-CD19 alone did not exhibit discernable cytotoxicity toward Raji or CD5⁺Raji cells at all concentrations tested and that DT-anti-CD5 was not toxic to Raji cells and exhibited only limited toxicity toward CD5⁺Raji cells at higher concentrations. However, the combination of DT-anti-CD5 and GrB-anti-CD19 fusion proteins was able to arrest protein synthesis in CD5⁺Raji cells with the EC50 of 423.3 pM, while the parental Raji B cell line was not sensitive to the same treatment (FIG. 3B). GrB-anti-CD19 activated DT-anti-CD5 in a dose-dependent manner (FIG. 4) and fully activated the engineered DT-anti-CD5 at about 1.0 nM, which is well below the concentrations where GrB alone exhibits apoptotic activity (Liu et al. Mol. Cancer Ther. 2(12):1341-50 (2003)). Together, these results demonstrate that DT-anti-CD5 can be targeted to CD5⁺ cell through anti-CD5 ScFv domain and can be activated efficiently by GrB-anti-CD19.

To address if the anti-CD19 ScFv domain of the GrB-anti-CD19 is required for efficient targeting of granzyme B activity to the target cells, we performed additional cytotoxicity assays using Jurkat and CD19⁺Jurkat cell lines. We found that CD19⁺Jurkat cells were much more sensitive to the combination of DT-anti-CD5 and GrB-anti-CD19 than Jurkat cells (FIG. 6A), indicating that DT-anti-CD5 was preferentially activated by GrB-anti-CD19 localized to the targeted CD19⁺Jurkat cell surface through CD19 binding interaction. The observed lower but significant cytotoxicity to Jurkat cells (CD19⁻) by these agents suggests that the targeted DT-anti-CD5 may be activated by free GrB-anti-CD19 in media. This hypothesis was confirmed by a separate experiment where both Jurkat and CD19⁺Jurkat cells were first treated with GrB-anti-CD19 at 4° C. for 30 min., and then washed with buffer to remove the unbound GrB-anti-CD19 from the media. Additional treatment with DT-anti-CD5 at 37° C. for 20 hours induced cytotoxicity in CD19⁺Jurkat cells, but not in Jurkat cells (FIG. 6B), indicating that the GrB-anti-CD19 bound to the CD19⁺Jurkat cells were responsible for DT activation. These results indicate that both anti-CD5 and anti-CD19 are necessary for selective killing of the target cells.

Pseudomonas Exotoxin (PEA) as the Cytotoxic Agent for Combinatorial Targeting

To broaden the scope of the combinatorial targeting strategy, we examined the use of a different bacterial toxin, Pseudomonas exotoxin A (PEA) in such a context. PEA intoxicates target cells in a manner similar to DT. Upon internalization through receptor-mediated endocytosis, PEA is cleaved by furin at the target cells. The ADP-ribosyl transferase domain is then translocated to cytosol assisted by the translocation domain of PEA and impairs protein translation machinery of the target cells by ADP-ribosylating elongation factor 2. We designed anti-CD5-PEA fusion protein based in part on a published strategy (Di Paolo C. et al., Clin. Cancer Res. 9:2837-48 (2003)), and additionally, replaced the furin cleavage site (RQPR↓SW) with a granzyme B cleavage sequence (IEPD↓SG) (FIG. 7A). The anti-CD5-PEA fusion protein was prepared by refolding the aggregated fusion proteins from bacterial inclusion body using a refolding protocol described by Umetsu M. et al. (J. Biol. Chem. 278:8979-8987 (2003)). The purified anti-CD5-PEA fusion protein was highly pure, as judged by Coomassie Blue staining of the refolded anti-CD5-PEA by SDS-PAGE (FIG. 7B). It is susceptible to proteolytic cleavage by mouse granzyme B, yielding expected products (FIG. 7C).

To evaluate the ability of anti-CD5-PEA to kill target cells, we performed cytotoxicity assays as described above. We found that anti-CD5-PEA alone was not toxic to either target (CD5⁺Raji and CD5⁺JVM3) or non-target (Raji and JVM3) cells (FIG. 8), and that αCD5-PEA selectively killed target cells (CD5⁺Raji and CD5⁺JVM3) only in the presence of the second component of combinatorial targeting agents, GrB-anti-CD19, with apparent EC50 of 1.07 nM and 0.81 nM for CD5⁺Raji and CD5⁺JVM3 cells, respectively (FIG. 8).

Identification and Characterization a PEA-Like Protein from Vibrio Cholerae TP Strain

In the course of studying anti-CD5-PEA, we identified a putative toxin (GenBank accession number-AY876053) found in an environmental isolate (TP strain) of Vibrio Cholerae (Purdy A. et al., J. of Bacteriology 187:2992-3001 (2005)). Although this putative Vibrio Cholerae Exotoxin (VCE) only shares moderate protein sequence homology to PEA (33% identities and 49% positives), the residues that are critical for the function of PEA are conserved in VCE, including the active site residues (H440, Y481, E553 in PE), a furin cleavage site in the domain II, and an ER retention signal at the C-terminus (FIG. 9). Furthermore, using molecular simulation tools the VCE catalytic domain sequence was successfully threaded onto the structure of the PEA catalytic domain, consistent with the notion that VCE folds into a structure similar to that of PEA and thus may possess a similar enzymatic activity (Yates S. P., TIBS 31:123-133 (2006)).

To test whether VCE is a PEA-like toxin, we constructed several anti-CD5-VCE synthetic genes and produced anti-CD5-VCE fusion proteins in E. coli following the expression and purification protocols for anti-CD5-PEA (FIG. 10B). Like anti-CD5-PEA, the anti-CD5-VCE fusion protein bearing a granzyme B site can be cleaved specifically at the granzyme B cleavage site by both mouse granzyme B and GrB-anti-CD19 fusion protein. We then tested the ability of anti-CD5-VCE to kill target cells in the presence or absence of GrB-anti-CD19 and found that, like DT-anti-CD5 and anti-CD5-PEA fusion proteins, anti-CD5-VCE fusion protein alone was not toxic to target cells, and only in the presence of GrB-anti-CD19 fusion protein it selectively killed target cells (FIG. 11).

Two unexpected advantages of VCE in comparison with PEA relate to expression in E. coli and activity. While anti-CD5-PEA could only be produced in E. coli in insoluble form, anti-CD5-VCE was solubly expressed in E. coli, allowing facile His-tag mediated column purification. In addition, in the presence of GrB-anti-CD19, anti-CD5-VCE showed higher specific toxicity to CD5⁺Raji cells than anti-CD5-PE. When cytotoxicity profiles of anti-CD5-VCE, anti-CD5-PEA, and DT-anti-CD5 to CD5⁺Raji cells were determined simultaneously, the relative potency illustrated by observed EC₅₀ values were: anti-CD5-VCE (˜1.3 nM)<DT-anti-CD5 (˜3.0 nM)<anti-CD5-PEA (˜4.8 nM). Since VCE and PEA can be predicted to share a similar translocation/intoxication mechanism due to their similar domain structures, it is surprising that VCE is significantly more toxic. The increased toxicity of VCE may be due to more efficient translocation of its ADP-ribosyltransferase by the VCE translocation domain, or the intrinsically higher activity of its ADP-ribosyltransferase. A synthetic toxin comprising the VCE translocation domain and the PEA ADP-ribosyltransferase domain is ˜300-fold less toxic to target cells than VCE toxin.

To further assess the efficacy of the combinatorial targeting strategy, we compared the cytotoxicity of three fusion proteins: the anti-CD5-VCE bearing a granzyme B cleavage site, the anti-CD5-VCE fusion protein with the endogenous furin cleavage site, and the anti-CD5-VCE fusion protein in which one of the active sites was mutated (glutamic acid 613 to alanine). As expected, the E613A active site mutation failed to kill target cells at all concentrations tested (FIG. 11). Although replacing the furin cleavage site with a granzyme B cleavage site substantially reduced the toxicity of anti-CD5-VCE fusion protein, the addition of 1.0 nM GrB-anti-CD19 fully restored its cytotoxicity (FIG. 11). These results clearly demonstrate that combinatorial targeting agents are not only highly selective, but also as effective as conventional immunotoxins.

N-terminal Growth Factor Like Domain of uPA (Urokinase-Like Plasminogen Activator) as a Targeting Mechanism for Combinatorial Targeting Strategy

Naturally occurring peptides has been shown to bind their cognate receptors with high selectivity and affinity. One of such examples is the binding of uPA to its receptor uPAR. It has been shown that the region of u-PA responsible for high affinity binding (K_(d)≈0.5 nM) to uPAR is entirely localized within the first 46 amino acids called N terminal growth factor like domain (N-GFD) (Appella E., et al., J. Biol. Chem. 262:4437 (1987)). To examine if naturally occurring protein sequences such as the N-GFD may be adapted to serve as a targeting principle for the combinatorial targeting strategy, we replaced the ScFv domain of anti-CD5-VCE fusion protein with N-GFD to produce N-GFD-VCE and tested its efficacy in selective killing uPAR⁺ cells in combination with the GrB-anti-CD19 fusion protein. We chose to use CD19⁺Jurkat cells for the cytotoxicity assay since it has been shown that Jurkat cells express a moderate level of uPAR and are sensitive to DTAT, a diphtheria toxin/urokinase fusion protein that targets uPAR⁺ cells (Ramage J. G. et al. Leukemia Res. 27:79-84 (2003)). We found that N-GFD-VCE bearing the native furin cleavage site is toxic to CD19⁺Jurkat cells, but not to u-PAR negative Raji cells, indicating that cell targeting selectively is achieved exclusively through the N-GFD domain of N-GFD-VCE. N-GFD-VCE fusion protein bearing a granzyme B site alone exhibited only limited toxicity at higher concentrations and was able to kill CD19⁺Jurkat cell line in the presence of GrB-anti-CD19 at concentrations where N-GFD-VCE itself was not toxic to the target cells (FIG. 12). These results demonstrate that a naturally occurring ligand can serve as targeting mechanism for combinatorial targeting.

Selective Killing of PBMNC from a CLL Patient Using the Combination of Anti-CD5-VCE and GrB-Anti-CD19

To test whether combinatorial targeting agents can specifically kill B cell-chronic lymphocytic leukemia cells, we carried out cytotoxicity assay with purified peripheral blood mononuclear cells (PBMNC) from a B-CLL patient. FACS analysis indicated that about 30% of PBMNC was CD5⁺ B cells (FIG. 13A). We found that each individual component of targeting agents was not toxic to PBMNC (FIGS. 13B and 13C). Furthermore, at the concentrations where combinatorial targeting agents arrested all the protein synthesis activity of the reported cell line (CD5⁺Raji), about 30% of total protein synthesis activity from PBMNC was arrested. Importantly, no more inhibition of protein synthesis was observed as we increased the concentration of DT-anti-CD5, consistent with the notion that the combinatorial targeting agents might only arrest protein synthesis activity of the target cell population, i.e., CD5⁺ B cells. Taken together, our data show that combinatorial targeting agents can be deployed to eliminate specific cell populations from heterogeneous mixtures of cells with minimal toxicity to other cell types.

H. Preparation of Anti-CD5-Aerolysin and Anti-CD19-Aerolysin Fusion Proteins

Gene Construction of Tagged, Modified Large Lobe of Aerolysin, Tagged Anti-CD5 ScFv, and Tagged Anti-CD19 ScFv

Aerolysin was amplified from the genomic DNA of Aeromonas hydrophila (ATCC: 7965D) using Faststart high fidelity PCR mix (Roche). The PCR product was digested with NcoI and XhoI and cloned into a pET22b (Novagen). The 3′ end of the clone was subsequently repaired by amplification and digested with NcoI and SalI and recloned into pET22b using NcoI and XhoI sites. There are many different variants of aerolysin and the sequence we obtained most closely resembled an aerolysin clone aer4 (GenBank: X65043). The most significant similarity between our clone and aer4 is in the activation peptide sequence separating the mature pore-forming toxin and the pro-peptide. This differs greatly from the sequence identified from the original aerA gene which is thought to be activated by furin (DSKVRRAR↓SVDG). The activation moiety of our clone was mutated from the native activation moiety (ASHSSRARNLS) to a sequence that could be recognized by human granzyme B (ESKGIEPD↓SGVEG) and tobacco etch virus protease TEV (ESKENLYFQ↓GVEG). We performed site specific mutagenesis using a Phusion polymerase based PCR mutagenesis method (New England Biolabs). These mutants were further modified to delete the small lobe of the native protein and replace it with a sortase substrate sequence (GKGGSNSAAS) using site directed mutagenesis. The resultant clones are referred to as GK-aerolysin_(GrB) and GK-aerolysin_(TEV), respectively.

Anti-CD5 ScFv was PCR amplified, each digested with NcoI and XhoI, and cloned into a pET28a (Novagen) variant modified to carry a sortase attachment signal LPETG upstream of the His-tag. anti-CD19 ScFv was PCR amplified, digested with NcoI and XhoI and cloned into a modified version of pET28a with a periplasmic signal sequence and a sortase attachment signal at the C-terminus.

Expression and Purification of Tagged Aerolysin Proteins, Tagged Anti-CD5 ScFv, and Tagged Anti-CD19 ScFv

GK-Aerolysin_(GrB) (FIG. 16) and GK-aerolysin_(TEV) were expressed in BL21 star cells at 25° C. after 0.2 mM IPTG induction for 5 hrs. Cells were pelleted and resuspended in lysis buffer (20 mM Tris pH 8, 150 mM NaCl, 0.3 M NH4Cl, 0.1% Triton X-100, 0.2 mg/mL lysozyme) and incubated for 1 hr at 4° C. This was followed by sonication to lyse the bacterial cells and the mixture was spun down and the supernatant was incubated with Ni-NTA agarose (Qiagen). The column was washed with HS buffer (20 mM Tris pH 8, 150 mM NaCl, 1 M NH4Cl, 0.1% Triton X-100) and 20 mM imidazole wash buffer (20 mM Tris pH 8, 150 mM NaCl, 20 mM imidazole) and eluted with 250 mM imidazole elution buffer (20 mM Tris pH 8, 150 mM NaCl, 250 mM imidazole). The protein was then dialyzed against 20 mM Tris pH 7.5 and 150 mM NaCl. Sortase A was purified using a similar protocol.

The ScFvs were expressed as insoluble inclusion bodies in BL21 cells. The inclusion bodies were isolated and then resuspended in redissolving buffer (5M GuCl, 20 mM Tris pH 8, 150 mM NaCl, 0.1% Triton X-100, 5 mM mercaptoethanol). The solution was sonicated to dissolve the protein and then mixed with 4 mL Ni-NTA slurry. The protein was purified under denaturing conditions in the presence of 5M GuCl, and eluted with imidazole (5 mM GuCl, 20 mM Tris pH 8, 150 mM NaCl, 250 mM imidazole, 5 mM mercaptoethanol). The protein was refolded using serial dialysis approach using differing amounts of GuCl and arginine (Umetsu M. et al. J. Biol. Chem. 278:8979-8987 (2003)). The refolded protein was finally dialyzed against 20 mM Tris pH 8, 150 mM NaCl.

Construction of Anti-CD5-Aerolysin and Anti-CD19-Aerolysin_(GrB) Using Sortase A Conjugation

S. aureus sortase A is expressed in soluble form from E. coli (Zong Y. et al. J. Biol. Chem. 279:31383 (2004)). Purified Sortase A was immobilized on agarose at approximate 10 mg/mL using aminolink plus coupling kit (Pierce). The GK-aerolysin proteins and the refolded scFvs were mixed at 1:2 ratio respectively and incubated with Sortase A-agarose in the presence of 0.1M Tris pH 9, 5 mM CaCl₂, 0.01% Tween-20, and incubated overnight at room temperature. The conjugation mix was filtered through a 0.2 micron spin filter and the mixture was purified on a Q-anion exchange column (GE Healthcare) to separate the conjugated aerolysin from the excess ScFv (FIG. 17C). The protein was concentrated and quantified by UV absorbance in preparation for cell based assays.

I. Cytotoxicity Assay (MTS Assay) of Aerolysin Based Toxin Fusions

Promega Cell Titer 96 Aqueous Non-radioactive Cell Proliferation Assay was used to determine cell viability. Cells were placed in a 96-well plate at 5 10⁴ cells per well in 90 μl RPMI with 10% calf serum (Hyclone, fortified with Fe²⁺). 10 μl of various concentrations of GrB-anti-CD19 ScFv and/or anti-CD5-Aerolysin_(GrB) fusion proteins were added to cells and incubated at 37° C. for 48 hours in 5% CO₂ incubator. MTS reagent (25 μl, Promega, G358A) was then added to each well and allowed to incubate for over 4 hours at 37° C. At the end of the incubation period, the A₄₉₀ was recorded using a SPECTRA max ELISA plate reader (Molecular Devices). Cell viability was normalized to control wells treated with protein storage buffer or 1 mM cycloheximide. The reported data represent the average readings from duplicate wells.

Anti-CD5-Aerolysin_(GrB) is Selectively Activated by GrB-anti-CD19

To investigate whether the engineered aerolysin fusion protein containing a GrB cleavage site and a CD5 binding moiety may be used as the toxin principle in the context of combinatorial targeting of CD5⁺/CD19⁺ cells, the cytotoxicity of anti-CD5-Aerolysin_(GrB) to CD5⁺Raji and CD19⁺Jurkat cells was assayed in the presence or absence of 2 nM of GrB-anti-CD19. As shown in FIG. 18, potent cytotoxicity is only observed when GrB-anti-CD19 is present, with EC₅₀≈0.3-0.4 nM and 6.5 nM to CD5⁺Raji and CD19⁺Jurkat cells, respectively. Virtually no toxicity was observed without the addition of GrB-anti-CD19. Such a low side effect by a aerolysin base protoxin may be attributable to its intoxication mechanism, which involves extracellular proteolytic activation followed by pore formation on cell surface (Howard and Buckley, J. Bateriol. 163:336-340 (1985)). In comparison, DT, PE, or VCE based protoxins are activated inside targeted cells during the translocation process (Ogata et al. J. Biol. Chem. 267:25396-25401 (1992)), during which some intracellular, endogenous proteolytic activities may cleave the heterologous protease cleavage site to activate them, albeit to much less extent than when activated specifically by a targeted activator.

Specific Anti-CD5 ScFv/CD5 Interaction at Cell Surface is Required for the Cytotoxicity of Anti-CD5-Aerolysin_(GrB)-Anti-CD19

The necessity of CD5 binding of anti-CD5-Aerolysin_(GrB) for cell targeting was confirmed by the fact that GK-Aerolysin_(GrB), which lacks the anti-CD5 ScFv domain, is not toxic to CD5⁺Raji cells under the conditions tested. The requirement for specific interaction between anti-CD5 ScFv and cell surface CD5 was further verified by the observation that anti-CD5-Aerolysin_(GrB), in combination to GrB-anti-CD19, is not toxic to Raji cells, which lack the CD5 surface marker (FIG. 18B). Although it is not surprising that a anti-CD5-scFV moiety could direct anti-CD5-Aerolysin_(GrB) fusion protein to CD5⁺Raji cells, it is not obvious that the anti-CD5-scFV moiety could simply replace the small lobe of aerolysin and successfully function as an integral part of aerolysin. The small lobe of the wild type aerolysin is known to recognize and specifically bind to N-glycans on GPI-anchored proteins, suggesting that it recognizes a site to which both the N-glycan and the GPI-glycan core contribute (MacKenzie et al. J. Biol. Chem. 274:22604-22609 (1999)). Conversely, domain 2 within the large lobe of aerolysin is thought to contribute to the binding of the GPI-core. The specific cytotoxicity to CD5⁺/CD19⁺ cells achieved by anti-CD5-Aerolysin_(GrB)/GrB-anti-CD19 demonstrated that the contribution of the small lobe to the binding of N-glycan and corresponding GPI-glycan core may be replaced by other interactions between a binder and the surface antigen it recognizes, and the surface marker does not have to be a GPI-anchored protein.

Cytotoxicity to CD5⁺JVM3 and Jeko-1 Cell Lines

JVM-3 is a cell line that has been used to establish a B-CLL-like xenograft mouse model (Loisel S. et al. Leuk. Res. 29:1347-1352 (2005)), even though it is CD5⁻. As described above, we have generated a CD5⁺JVM3 cell line to test combinatorial targeting agents. Jeko-1 cell line is a mantle cell lymphoma cell line that is CD5⁺/CD19⁺ (Jeon et al. Brit. J. Haematol. 102:1323-1326 (1998)). Potent cytotoxicity of anti-CD5-Aerolysin_(GrB) to these cells is observed in the presence of 2 nM of GrB-anti-CD19 (FIG. 19), with estimated EC₅₀ of 2.1 nM and 22.4 nM, respectively. Since Jeko-1 cells naturally possess both CD5 and CD19 surface antigens, these data illustrate that combinatorial targeting reagents are capable of selectively destroying cancer cells by recognition of cell surface targets present on the cell surface at native levels.

Construction and Expression of Wild Type and Mutant DT Fusion Proteins Bearing Phosphorylation Sites that Block Furin Cleavage when Phosphorylated

The gene encoding full length DT (synthesized by Genscript Corporation) was cloned into pBAD102/D-TOPO (Invitrogen Corporation). Single amino acid insertion at the furin cleavage site was achieved using a site-directed mutagenesis kit from Stratagene (QuikChange® 11 Site-Directed Mutagenesis Kit). The original enterokinase recognition sequence in the vector plasmid was changed to a TEV protease recognition sequence using PCR.

All plasmid constructs were transformed into One Shot® TOPO10 competent cells (Invitrogen Corporation). Positive colonies were selected. For protein induction, a single positive bacterial colony was inoculated into 2 ml of LB and transferred into 100 ml LB after overnight incubation. After OD reached 0.6, the culture was moved to 16° C. incubator, to which was added arabinose to a final concentration of 20 ppm and the induction lasted at least for 4 hours. Bacteria were precipitated at 2000 g for 10 minutes and the cell pellet was then suspended in 8 ml buffer of 25 mM NaH₂PO₄, 250 mM NaCl at pH 8.0. The cell solution was then incubated with 8 mg of lysozyme on ice for 30 minutes. After sonication, the lysate was centrifuged at 3,000 g for 15 minutes, and the resulting supernatant was purified by Ni-NTA agarose purification following manufacturer's recommended procedures (Invitrogen Corporation).

After purification, the protein solutions were dialyzed against a buffer of 25 mM Tris, 250 mM NaCl and 10% glycerol at pH 7.5 for overnight, to provide a buffer system that is compatible with furin cleavage and phosphorylation reactions. All the fusion proteins made (DT, DT^(A), DT^(S), DT^(AT)) are depicted in FIG. 21 with the corresponding furin cleavage sites shown.

Phosphorylation of Fusion Proteins

To examine the efficiency and specificity of site-specific phosphorylation of Trx-DT fusion proteins DT, DT^(A), DT^(S), and DT^(AT), a number of commercially available kinases were screened. Protein kinase A (PKA) was identified as the most efficient for these fusions. Phosphorylation reaction was carried out in 20 μl of 50 mM Tris-HCl/10 mM MgCl₂ pH 7.5 buffer containing 1 μg of protein, 1 μl of protein kinase A, and 2 μl of 1 mM ATP (New England Biolabs). The mixture was incubated at 30° C. for 20 minutes. In order to visualize the phosphorylation product, in some phosphorylation experiments ATP was supplemented with γ-³²P-ATP (3000 Ci/mmol, Perkin Elmer Life and Analytical Science) to yield ³²P labeled Trx-DT. It was found that PKA adds the radioactive phosphate group to all the fusion proteins, producing a single product as shown by SDS-PAGE analysis (FIG. 22B, top panel). The labeling efficiency of the Trx-DT fusions, which corresponds to phosphorylation efficiency, is found to be DT^(A)>DT^(S)>DT^(AT)≈DT.

Furin Cleavage of Trx-DT and Phosphorylated Trx-DT Fusion Proteins

To analyze whether the phosphorylatlon at furin cleavage site within the Trx-DT fusion proteins have any effect on furin cleavage efficiency, the unlabeled and phosphate-labeled fusion proteins were incubated with furin at 37° C. For each furin digestion, 2 μg of protein was mixed with 2 units of furin (New England Biolabs) in a total reaction volume of 20 μl at 37° C. Reaction buffer contained 100 mM Tris-HCl, 0.5% Triton X-100, 1 mM CaCl₂ and 0.5 mM dithiothreitol at pH 7.5. The reaction mixtures were analyzed by SDS-PAGE using the samples without turin treatment as controls. We found that the control samples contained some nicked products of 35 kD and 41 kD, which are consistent with fragmentation at the furin cleavage site. This phenomenon has been observed by others previously and is considered the result of undesired proteolytic cleavage during protein purification. After a 20 minute furin treatment, the DT, DT^(A), DT^(S), and DT^(AT) samples showed substantially more cleavage products of 35 kD and 41 kD (FIG. 21B), demonstrating site specific cleavage of non-phosphorylated samples, as expected. However, the phosphorylated proteins pDT^(A), pDT^(S), and pDT^(AT) showed reduced sensitivity to furin cleavage. While significant digestion on pDT could be observed after one hour, no obvious digestion could be observed for pDT^(A), pDT^(S), and pDT^(AT). The digestion was then continued for overnight. After furin treatment for 20 hours, the cleavage of pDT was near completion, but only about 5%, 10%, and 50% of pDT^(A), pDT^(AT) and pDT^(S) were fragmented, respectively (FIG. 22B). The significantly reduced lability of pDT^(A), pDT^(AT) and pDTs to furin due to phosphorylation suggests that they may potentially be used as protoxins which are activated by dephosphorylation to provide a natively activatable toxin, i.e. one that can be activated by endogenous furin/kexin-like proteases.

Preparation of DT^(A)-Anti-CD19 and pDT^(A)-Anti-CD19 Fusion Proteins

The Trx-DTA-anti-CD19 fusion gene containing an alanine insertion at furin cleavage site ₁₉₀RVRR↓ASV₁₉₅ was constructed by subcloning from the corresponding Trx-DT (DT^(A) in FIG. 21A) and DT_(GrB)-anti-CD19 fusion genes. Trx-DTA-anti-CD19 fusion protein was expressed in E. coli and the soluble fraction was collected and purified using standard His-tag purification. The purified Trx-DT^(A)-anti-CD19 was treated with TEV protease to remove the Trx tag and afford DT^(A)-anti-CD19.

The purified DT^(A)-anti-CD19 was further phosphorylated using PKA and ATP using the procedure described above to generate pDT^(A)-anti-CD19 (FIG. 22A).

Dephosphorylation of pDT^(A)-Anti-CD19

Fusion protein pDTA-anti-CD19 was treated with recombinant protein phosphatase 2C (PP2C) produced in E. coli, and its dephosphorylation was observed by SDS-PAGE. The resulting DT^(A)-anti-CD19 contains the RVRR↓AS sequence, which is activatable by furin that is present in mammalian cells. PP2C was selected for the dephosphorylation because it has been shown that it can remove the phosphate group on RRAT^(P)VA or RRAS^(P)VA efficiently (Deana et al., Biochim. Biophy. Acta, 1051:199-202 (1990)), which are very similar to the modified furin cleavage site within pDT^(A)-anti-CD19.

Cytotoxicity Assay of D-Anti-CD19 and pDT^(A)-Anti-CD19 Fusion Proteins

Both DT^(A)-anti-CD19 and pDT^(A)-anti-CD19 were tested by protein synthesis inhibition cytotoxicity assay as described above, using cells that contain both the CD5 and CD19 surface antigens, i.e. Jeko-1, CD5⁺JVM3, CD5⁺Raji, and CD19⁺Jurkat cells. Various concentrations of DT^(A)-anti-CD19 and pDT^(A)-anti-CD19 were tested, and a positive inhibition control was provided by adding cycloheximide to each cell line. The results (FIG. 23B) show that the unphosphorylated DT^(A)-anti-CD19 fusion is very toxic to all the cells tested, with IC50˜0.01-0.1 nM; whereas the phosphorylated pDTA-anti-CD19 fusion is not toxic to these cells under similar conditions.

These results demonstrate that it is feasible to establish a protoxin activation strategy, in which the proactive moiety (e.g., furin cleavage site RVRR↓AS) within a protoxin (e.g., DT^(A)-anti-CD19) is masked by a chemical modification (e.g., phosphorylation at the Serine) to afford a protoxin (e.g., pDT^(A) anti-CD19−); the protoxin may be converted by an activator (e.g., phosphatase PP2C) to a natively activatable toxin (e.g., DT^(A)-anti-CD19), which is activated by furin activity natively present in mammalian cells.

This strategy should be applicable to any protoxin that may be naturally activated by intracellular or extracellular proteolysis. Examples of such toxins include but not limited to, ADP-ribosylating toxin such as DT, PE, and VCE, pore-forming toxin such as aerolysin and Clostridium perfringens ε-toxin, pro-RIP toxin such as pro-ricin, and zymogen-based toxin such as pro-GrB. Examples of enzyme activities that may be used to modify/demodify as protoxin modifying reagent and protoxin proactivator include but are not limited to, kinases and phosphatases for phosphorylation and dephosphorylation, respectively; O-GlcNAc transferase and O-GlcNAcase for glycosylation and deglycosylation, respectively; and E1/E2 and Senp2 for sumoylation and desumoylation, respectively.

Production of Mature GrB-(YSA)₂ and Protease Activatable Pro-GrB-(YSA)₂

In CTLs and NK cells, GrB is initially expressed as an inactive precursor protein. This pre-pro-GrB carries an N-terminal signal peptide that directs packaging of the protein into secretory granules. The enzymatic activity of GrB is strictly controlled by the activation dipeptide Gly-Glu, which is cleaved by dipeptidyl peptidase/cathepsin C during transport into storage vesicles. We have constructed recombinant GrB in a pro form, which may be matured either by a separate step of proteolytic removal of the extra residues located N-terminal to the first residue Ile of GrB, or by in situ activation conferred by a natively present protease in the host cells.

As shown in FIG. 20A, two pro-GrB-(YSA)₂ fusion proteins were designed and constructed, an enterokinase activatable DDDDK-GrB-(YSA)₂ fusion protein, and a furin activatable RSRR-GrB-(YSA)₂ fusion protein. DDDDK-GrB-(YSA)₂ was produced by transfecting 293T cells with plasmids expressing this fusion protein. The pro-enzyme was produced as a secreted form and was first purified with Ni affinity chromatography. Purified DDDDK-GrB-(YSA)₂ was activated by adding enterokinase in vitro. Using a fluorogenic peptide (Ac-IEPD-AMC), it was demonstrated that the enzymatically active GrB-(YSA)₂ was obtained by proteolytically cleaving the sequences N-terminal to the naturally matured GrB sequence (amino acid 21 to 247) using added enterokinase, which recognizes and cleaves at DDDDK↓ (FIG. 20B).

On the other hand, GrB-(YSA)₂ may be isolated in its mature form in 293T cells directly if the fusion construct is designed to be activated by furin, which is naturally present in mammalian cells. Supernant of 293T cells transfected with plasmids expressing RSRR-GrB-(YSA)₂ was collected and the activity of GrB was comparable to that of GrB-(YSA)₂, which was activated in vitro by enterokinase treatment of DDDDK-GrB-(YSA)₂.

These experimental results demonstrate that the status of GrB activity may be manipulated by either exogenbus (e.g., enterokinase) or endogenous (e.g., furin) proteolytic activities. Such controlled activation is particularly useful for the combinatorial targeting described in the present invention. For example, the activation of DT_(GrB)-anti-CD5 protoxin fusion may only be achieved when the targeted cells are also bound to the DDDDK-GrB-(YSA)₂ fusion, where the exogenous enterokinase is introduced by a cell-targeting moiety recognizing a third cell surface target. On the other hand, in many mammalian cells the availability of RRSR-GrB-(YSA)₂ fusion is sufficient to be activated DT_(GrB)-anti-CD5 protoxin fusion because these cells natively expresses furin, which can activate proactivator RRSR-GrB-YSA.

J. Targeting Breast Cancer Cells Using Surface Marker EphA2 and Claudin3/4

In one particular example, the protoxin and protoxin activator fusion proteins of the invention were directed towards breast cancer cells expressing EphA3 and claudin3/4.

Construction of a DT_(GrB)-CCPE Fusion Gene

The translocation domain and catalytic domain of DT from the DT_(GrB)-anti-CD5 gene was cloned into pBAD/D-TOPO-vector (Invitrogen) that contains a His-Patch Thioredoxin. A factor Xa site was also introduced directly upstream of the DT to provide an opportunity to later remove the thioredoxin front the fusion protein. The gene encoding C-CPE was synthesized (Genscript Corporation). The C-CPE insert containing a polyhistidine tag (H6) at C-terminus was ligated into the pBAD/D-TOPO-DT vector described above to generate the fusion gene. A TEV protease cleavage site was introduced using PCR based mutagenesis and Phusion™ High-Fidelity DNA Polymerase (New England Biolabs). The recognition site used was ELNYFQ↓G, and replaced the Factor Xa site (I-E-G-R) in the original construct.

Expression of DT_(GrB)-CCPE

A one liter culture of E. coli containing the pBAD/D-TOPO-Trx-DT_(GrB)-CCPE plasmid was grown to OD600=0.6 in LB containing ampicillan. The culture was induced with 0.02% arabinose at 18° C. overnight. Fusion protein was purified using Ni-NTA agarose resin (Qiagen) and dialyzed against PBS.

TEV protease was used to remove the thioredoxin from the Trx-DT_(GrB)-CCPE construct. The DT_(GrB)-CCPE was purified from the TEV protease and the thioredoxin using an amylose resin column (New England Biolabs) followed by a Ni-NTA agarose column (Qiagen). The purified protein was dialyzed against PBS.

Construction of GrB-(YSA)₂ Gene Fusion

A twelve residue peptide, YSA, having the sequence YSAYPDSVPMMS, has been reported to be a specific binder to EphA2 receptors (Koolpe, et al. J Biol Chem. 280:17301-11 (2005)), which are overexpressed in number of cancers. A DNA encoding the fusion of two YSA peptides was synthesized and cloned into pIC9 vector along with the GrB gene in a 3-piece ligation reaction. The resulting plasmid was confirmed to contain the desired GrB-(YSA)₂ DNA, which was then sub-cloned into pEAK15-GrB-CD19L vector that was used for mammalian expression of the GrB-anti-CD19 fusion discussed above. The pEAK15-GrB-(YSA)₂ construct contains a leader sequence for secretion of the expressed protein, as well as an enterokinase site directly upstream of the Granzyme B.

Expression and Purification of GrB-(YSA)₂

The pEAK15-GrB-(YSA)₂ plasmid was transfected into 293ETN cells using TransFectin™ Lipid Reagent (BioRad) following recommended procedure. Cells were incubated for 2 days in OptiMEM (Gibco), and the supernatant was collected. The secreted protein was purified from media supernatant using Ni-NTA resin (Qiagen), then dialyzed against Tris-Cl buffer.

The purified pro-GrB-(YSA)₂ was incubated with Enterokinase to remove the leader sequence and flag-tag from N-terminal side of Granzyme B. Thus activated GrB-(YSA)₂ was then separated from the signal peptide using Ni-NTA resin (Qiagen), to be used to activate DT_(GrB)-CCPE fusion (FIG. 24).

This system again exemplifies an activation sequence that involves three elements, enterokinase, pro-GrB-(YSA)₂, and DT_(GrB)-CCPE, with the end result of DT activation at the cells targeted by C-CPE and YSA. It is anticipated a triple-component activation cascade may be established by using an enterokinase that is linked to a cell-targeting moiety that recognizes a third surface antigen. For example, in order to target certain breast cancer cells, EpCAM may be used as the third surface marker (targeted by an anti-EpCAM scFv) for enterokinase, in combination with claudin3/4 (targeted by C-CPE) and EphA2 (targeted by multimerized YSA or anti-EphA2 scFv).

Cytotoxicity of Protoxin DT_(GrB)-CCPE Activated by GrB

Protoxin DT_(GrB)-CCPE fusion protein was activated in vitro using mouse GrB (Sigma) prior to exposing it to cells. Equal numbers of HT-29 cells, which express Claudin-3/-4, were seeded in a 96 well plates and allowed to settle for 24 hours. Activated DT_(GrB)-CCPE was added directly to the wells in concentrations ranging from 0.03 nM up to 0.6 μM, each concentration in triplicate. Cycloheximide was used as a cell growth inhibition control, and PBS was added to wells as a buffer control. Cells were incubated in the presence of the activated DT_(GrB)-CCPE fusion for 48 hours, and cytotoxicity was then measured with CellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS) (Promega) as outlined in product manual. Results were analyzed using GraphPad Prism 4.

K. Multistep Synthesis of Branched Chemical Linker JL10

The invention features the use of branched chemical linkers between the various domains of the protoxin and protoxin activator fusion proteins. An example of the synthesis of one such linker is described below.

Synthesis of 14-amino-5-oxo-3,9,12-trioxa-6-azatetradecan-1-oic acid (JL01)

To a solution of 2,2′-(ethane-1,2-diylbis(oxy))diethanamine (1.4830 g, 10.0 mmol) in CH₃CN (15 mL) was added dropwise a solution of 1,4-dioxane-2,6-dione (1.1560 g, 10.0 mmol) in CH₃CN (5 mL) over 5 minutes and the mixture was stirred for 5 hours at room temperature. A colorless supernatant was discarded by decantation. 5 mL of CH₃CN was added and the mixture was vortexed for 30 seconds. The supernatant was decanted. The remaining residue was dissolved in 1M HCl (20 mL) and chromatographed with Dowex 50W 8 ion-exchange resin (15 mL resin, H⁺-form). The mono-acid product was eluted with water and followed by 0.15M of NH₄OH. The reaction afforded 37% yield of the mono-acid product as light yellow gum (JL01). ¹H-NMR (400 MHz, DMSO-d₆) δ_(H) 9.69 (t, J=5.20 Hz, 1H), 8.27 (br, 3H), 3.87 (s, 2H), 3.73 (s, 2H), 3.66 (t, J=5.40 Hz, 2H), 3.58 (m, 2H), 3.53 (m, 2H), 3.48 (t, J=5.00 Hz, 2H), 3.27 (m, 2H), 2.91 (t, J=5.40 Hz, 2H); ¹³C-NMR (101 MHz, DMSO-d₆) δ_(C) 174.17, 170.48, 72.94, 72.27, 69.89, 69.81, 69.39, 66.98, 48.63, 38.54; MS (ESI) m/z 265 (M⁺).

Synthesis of 14-(tert-butoxycarbonylamino)-5-oxo-3,9,12-trioxa-6-azatetradecan-1-oic acid (JL02)

To a solution of 14-amino-5-oxo-3,9,12-trioxa-6-azatetradecan-1-oic acid (0.9650 g, 3.7 mmol) in water (10 mL) was added NaHCO₃ (0.3739 g, 4.4 mmol) and the mixture was stirred at room temperature for 10 minutes. A solution of Boc₂O (0.9834 g, 4.5 mmol) in dioxane (5 mL) was added to the mixture and stirred at room temperature for overnight. The reaction crude was concentrated under reduced pressure. The residue was re-dissolved in water and washed with diethyl ether. The ether layer was discarded and the residue was acidified with 1M HCl and extracted with ethyl acetate. The organic layer was saved and dried over Na₂SO₄. After ethyl acetated was removed under reduced pressure, a pale yellow gum was obtained as product (JL02) in 1.3111 g. ¹H-NMR (400 MHz, DMSO-d₆) δ_(H) 12.79 (brs, 1H), 7.81 (t, J=5.80 Hz, 1H), 6.80 (t, J=5.40 Hz, 1H), 4.10 (s, 2H), 3.96 (s, 2H), 3.49 (s, 4H), 3.43 (t, J=5.80 Hz, 2H), 3.36 (t, J=6.00 Hz, 2H), 3.26 (m, 2H), 3.05 (m, 2H), 1.37 (s, 9H); ¹³C-NMR (101 MHz, DMSO-d₆) δ_(C) 171.43, 168.83, 155.62, 77.63, 70.03, 69.51, 69.49, 69.21, 68.87, 67.48, 38.06, 28.26.

Synthesis of ethyl 21,21-bis((3-ethoxy-3-oxopropoxy)methyl)-2,2-dimethyl-4,15,19-trioxo-3,8,11,17,23-pentaoxa-5,14,20-triazapentacosane-25-carboxylate (JL04)

Compound JL02 (1.2540 g, 3.44 mmol) and N-hydroxysuccinimide (0.5140 g, 4.47 mmol) were dissolved in CH₂Cl₂ (10 mL) and DMF (5 mL). The mixture was stirred at room temperature and a solution of DCC (0.8020 g, 3.88 mmol) in CH₂Cl₂ (10 mL) was added. The mixture was stirred for overnight and the white precipitates were removed by filtration. The filtrate was concentrated under reduced pressure to afford NHS ester. The NHS ester was re-dissolved in DMF and stirred in ice bath. After addition of a solution of amino triethyl ester^(JL05) (JL03, 1.5520 g, 3.68 mmol) in DMF (5 mL), the ice bath was removed and the mixture was stirred at room temperature for 63 hours. The reaction crude was filtered, washed with ethyl acetate and concentrated under reduced pressure. The residue was purified on silica gel column and afforded pale yellow gum product (JL04) in 94% yield. ¹H-NMR (400 MHz, DMSO-d₆) δ_(H) 10.57 (brs, 1H), 8.01 (t, J=5.60 Hz, 1H), 6.77 (t, J=5.40 Hz, 1H), 4.05 (q, J=7.20 Hz, 6H), 3.92 (s, 2H), 3.87 (s, 2H), 3.59 (t, J=6.00 Hz, 6H), 3.56 (s, 6H), 3.49 (s, 4H), 3.43 (t, J=6.00 Hz, 2H), 3.36 (t, J=6.20 Hz, 2H), 3.26 (m, 2H), 3.05 (m, 2H), 2.49 (t, J=6.40 Hz, 6H), 1.37 (s, 9H), 1.18 (t, J=7.20 Hz, 9H); ¹³C-NMR (101 MHz, DMSO-d₆) δ_(C) 172.84, 171.06, 168.75, 168.69, 155.61, 77.61, 70.29, 70.20, 69.51, 69.48, 69.20, 68.89, 68.14, 66.54, 59.89, 59.80, 59.39, 38.12, 34.52, 28.24, 25.25, 14.10.

Synthesis of 21,21-bis((2-carboxyethoxy)methyl)-2,2-dimethyl-4,15,19-trioxo-3,8,11,17,23-pentaoxa-5,14,20-triazapentacosane-25-carboxylic acid (JL06)

To a solution of compound JL04 (2.2230 g, 2.90 mmol) in THF (30 mL) was added 1M NaOH aqueous solution (15 mL). The mixture was stirred at room temperature for overnight and THF was removed under reduced pressure. The aqueous solution was acidified with 6M HCl to pH 2 and extracted with CH₂Cl₂. The organic layer was saved and dried over Na₂SO₄. Pale yellow gum was obtained as product (JL06) in 76% yield. ¹H-NMR (400 MHz, DMSO-d₆) ⁸ _(H) 12.14 (s, 1H), 8.00 (t, J=5.74 Hz, 1H), 7.05 (s, 1H), 6.75 (t, J=5.52 Hz, 1H), 3.91 (s, 2H), 3.86 (s, 2H), 3.56 (m, 12H), 3.47 (s, 4H), 3.41 (t, J=6.04 Hz, 2H), 3.35 (t, J=6.11 Hz, 2H), 3.25 (q, J=5.87 Hz, 2H), 3.04 (q, J=5.97 Hz, 2H), 2.40 (m, 6H), 1.89 (s, 2H), 1.35 (s, 9H); MS (ESI) m/z 772 ([M+4Na−3H]⁺), 726 ([M+2Na−3H]⁻).

Synthesis of 1-azido-2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethane (JL07)

To a solution of 1-chloro-2-(2-(2-(2-chloroethoxy)ethoxy)ethoxy)ethane (13.2310 g, 57.2 mmol) in DMF (100 mL) and water (20 mL) was added NaN₃ (11.353 g, 175 mmol) and the mixture was stirred at 80° C. for 40 hours. The filtrate was saved after filtration and concentrated under reduced pressure. The white slurry was diluted with ethyl acetate and hexanes (v/v 1:1, 200 mL) and the precipitates were removed by filtration. The filtrate was saved and washed with water (30 mL), brine (30 mL) and dried over Na₂SO₄. Pale yellow liquid was obtained as product (JL07) in 99% yield. ¹H-NMR (400 MHz, CDCl₃) δ_(H) 3.68 (m, 12H), 3.39 (t, J=5.05 Hz, 4H).

Synthesis of 2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethanamine (JL08)

To a solution of compound JL07 (14.4 g, ˜57.2 mmol) in ethyl acetate (45 mL) and diethyl ether (45 mL) was added 5% HCl (60 mL), followed by addition of Ph₃P (14.04 g, 53.5 mmol) and the mixture was stirred in ice-bath for over 1 hour. Then the ice-bath was removed and the reaction mixture was stirred at room temperature for 14 hours. The reaction crude was transferred to separatory funnel and the organic phase was removed. The aqueous phase was washed with ethyl acetate and cooled in ice-bath. 1M NaOH was added to adjust pH to 13. The product was extracted into CH₂Cl₂ and dried over Na₂SO₄. Pale yellow liquid was obtained as product (JL08) in 82% yield. ¹H-NMR (400 MHz, CDCl₃) δ_(H) 3.67 (m, 8H), 3.63 (m, 2H), 3.51 (t, J=5.23 Hz, 2H), 3.39 (t, J=5.07 Hz, 2H), 2.87 (t, J=5.21 Hz, 2H), 1.62 (s, 2H).

Synthesis of tert-butyl 33-azido-16,16-bis(17-azido-5-oxo-2,9,12,15-tetraoxa-6-azaheptadecyl)-10,14,21-trioxo-3,6,12,18,25,28,31-heptaoxa-9,15,22-triazatritriacontylcarbamate (JL09)

To a solution of compound JL06 (0.1367 g, 0.2 mmol) in CH₂Cl₂ (4 mL) was added a solution of compound JL08 (0.2619 g, 1.2 mmol) in CH₂Cl₂ (4 mL), followed by addition of DIEA (209 μL, 1.2 mmol), and the mixture was stirred at room temperature. A solution of DEPC (182 μL, 1.2 mmol) in CH₂Cl₂ (4 mL) was added dropwise into above mixture over 1 minute and still stirred at room temperature for overnight. After removal of solvent under reduced pressure, the residue was purified on silica gel column to afford 0.2047 g (80% yield) product JL09 as pale yellow liquid. ¹H-NMR (400 MHz, CDCl₃) δ_(H) 7.54 (br, 1H), 7.04 (br, 1H), 6.80 (br, 1H), 5.26 (br, 1H), 4.06 (s, 2H), 3.98 (s, 2H), 3.67 (m, 48H), 3.55 (m, 12H), 3.45 (t, J=5.30 Hz, 6H), 3.41 (t, J=4.97 Hz, 6H), 3.32 (br, 2H), 2.42 (t, J=5.81 Hz, 6H), 1.44 (s, 9H).

Synthesis of tert-butyl 33-amino-16,16-bis(17-amino-5-oxo-2,9,12,15-tetraoxa-6-azaheptadecyl)-10,14,21-trioxo-3,6,12,18,25,28,31-heptaoxa-9,15,22-triazatritriacontylcarbamate (JL10)

A solution of compound JL09 (0.2047 g, 0.16 mmol) in MeOH (0.64 mL) was added to a 2-neck 50 mL flask. 2 vacuum/Ar cycles were proceeded to replace the air in the flask with Ar. After quick addition of Pd/C to the flask, 2 vacuum/H₂ cycles were proceeded to replace Ar with H₂. The reaction mixture was vigorously stirred at room temperature under 1 atm H₂ pressure (balloon) for 72 hr. Pd/C was filtered off and pale yellow gum was obtained under reduced pressure as product (JL10, 0.1915 g) in 99% yield.

Preparation of JL10-(YSA)₂ and Removal of Protection Groups

To a solution of compound JL10 (0.1206 g, 0.1 mmol) in CH₂Cl₂ was added a solution of 0.6 mmol of N-terminus- and side-chain-protected YSA peptide in CH₂Cl₂, followed by addition of DIEA (105 μL, 0.6 mmol), and the mixture was stirred at room temperature. A solution of DEPC (91 μL, 0.6 mmol) in CH₂Cl₂ was added dropwise into above mixture over 1 minute and stirred at room temperature for overnight. After removal of solvent under reduced pressure, the residue was purified by chromatography. The protection groups were removed by sequential treatments of DEA (to remove base labile protecting groups) and TFA (to remove acid-labile protecting groups) and the resulting conjugate is ready for enzymatic ligation reaction.

Preparation of GrB-(YSA)₃

Granzyme B fusion proteins with a C-terminal tag LPETG or a LLQG tag are constructed and prepared using methods described above. Each GrB fusion was mixed with fully deprotected JL10-(YSA)₃ mixed at 1:2 ratio respectively and incubated with Sortase A-agarose in the presence of 0.1 M Tris pH 9, 5 mM CaCl₂, 0.01% Tween-20, and incubated overnight at room temperature. Each conjugation mixture was concentrated using a low MW cutoff spin concentrator, followed by extensive washing with buffer to remove excess JL10-(YSA)₃. The conjugate may be further purified using column choromatography. The resulting fusion protein possesses three YSA peptides with exposed N-terminus, as well as the GrB moiety in its active form with the exposed N-terminus (FIG. 24).

Because it is often challenging to discover short peptides that can bind to their cell surface targets with as high an affinity as antibodies, scFvs, or other scaffold-based binders, it may be necessary to multimerize these peptides. Whereas direct, repeated fusion of these peptides with flexible spacers is a convenient strategy for potentially synergistic binding, it does not allow the accessibility to the N-terminus or C-terminus of each peptide motif that is internally located. Since during phage display selection, multiple copies of peptides or proteins are displayed in a configuration that exposes their N-terminus (Kehoe and Kay, Chem. Rev. 2105(11):4056-72 (2005)), the selected peptides or proteins may be the most effective if similar structure is maintained in the targeting agents utilizing them. The use of branched chemical linkers such as described here provides an opportunity to display multiple peptides in any orientation with Respect to the fusion partner, which is critical for the GrB activity and may also be important for YSA-EphA2 interaction.

Construction and Expression of DT_(GrB)-Anti-CD2219 and GrB-Anti-CD1919

It has been reported previously that a bispecific scFv fusion protein, DT2219, was assembled consisting of the catalytic and translocation domains of diphtheria toxin fused to two repeating sFv subunits recognizing CD19 and CD22. DT2219 was shown to have greater anticancer activity than monomeric or bivalent immunotoxins made with anti-CD19 and anti-CD22 scFv alone and it showed a higher level of binding to patient leukemia cells and to CD19⁺CD22⁺ Daudi or Raji cells than did anti-CD19 and anti-CD22 parental monoclonal antibodies (Vallera et al., Clin. Cancer Res. 11(10):3879-88 (2005)). We similarly designed a protoxin DT_(GrB)-anti-CD2219 and GrB-anti-CD1919 to enhance the binding to targeted B-CLL cells, which are CD19⁺/CD22⁺. Whereas GrB-anti-CD1919 is expected to increase B cell affinity by simple synergistic binding of two binding motifs, DT_(GrB)-anti-CD2219 is designed to also take advantage of both CD19 and CD22 populations on the CD19⁺/CD22⁺ B cells.

FIG. 25 shows the schematic depictions of DT_(GrB)-αCD2219 and GrB-αCD1919 fusion proteins. DT-anti-CD2219 was secreted expressed from Pichia KM71. The endogenous furin cleavage site of the DT gene is replaced by a granzyme B cleavage site (IEPD↓SG). The toxin moiety and anti-CD5 ScFv are linked via a (G₄S)₃ linker (L). The two ScFv moieties were linked through HMA tag (Vallera et al., Clin. Cancer Res. 11(10):3879-88 (2005)). The secretion expression of GrB-anti-CD1919 was from 293 ETN. The configuration of GrB-anti-CD1919 is same as GrB-anti-CD19, except that an extra anti-CD19 ScFv moiety was fused to GrB-anti-CD19 via G₄ linker. In out cytotoxicity experiments, GrB-anti-CD1919 when combined with DT_(GrB)-anti-CD5 showed slightly higher selective toxicity to CD19⁺Jurkat cells than GrB-anti-CD19.

Preparation of NGFD-VCE_(TEV) and Anti-CD5-TEV

To provide another example of protease activator, NGFD-VCE_(TEV) was constructed from NGFD-VCE by replacing the endogenous furin cleavage site by TEV cleavage site (ENLYFQ↓G), and then expressed using similar procedures. The preparation of anti-CD5 scFv targeted TEV was accomplished using S. aureus Sortase A catalyzed ligation, because each moiety was optimally expressed under different conditions, i.e., periplasmic and cytoplamic expressions in E. coli, respectively. As illustrated in FIG. 26, LPETG-tagged anti-CD5 scFv was conjugated to GKGG-tagged TEV using standard Sortase A ligation procedures.

Proteolytic Activation of NGFD-VCE_(TEV) and Cytotoxicity Assay

As shown in FIG. 27A, the NGFD-VCE_(TEV) fusion protein, although not completely purified, was a substrate of recombinant TEV (Invitrogen) and was cleaved to generate a fragment of expected size. FIG. 27B shows the cytotoxicity assay results using CD19⁺Jurkat cells. When used in combination, 15 nM of NGFD-VCE_(TEV) and 1.5 nM of anti-CD5-TEV inhibited protein synthesis much more effectively than each reagent was used alone at the same concentrations. The observed synergistic effect of the two reagents demonstrates that NGFD-VCE_(TEV) is selectively activatable by anti-CD5-TEV on the same cell.

Cleavage of VCE

Polynucleotide and amino acid sequences for the constructs and proteins described above are set forth in Table 3.

SEQ ID NO: NAME NOTES SEQUENCE 74 VCE gi|58615288|gb|AAW80252.1| hypothetical exotoxin A [Vibrio cholerae] Wild type MYLTFYLEKVMKKMLLIAGATVISSMAHPTFAVEDELNIFDECRSPCSLTPEPGKPIQSKLSIPSDV sequence VLDEGVLYYSMTINDEQNDIKDEDKGESIITIGEFATVRATRHYVNQDAPFGVIHLKITTENGTKTY SYNRKEGEFAINWLVPIGEDSPASIKISVDELDQQRNIIEVPKLYSIDLDNQTLEQWKTQGNVSFSV TRPEHNIAISWPSVSYKAAQKEGSRHKRWAHWHTGLALCWLVPMDAIYNYITQQNCTLGDNWFGGSY ETVAGTPKVITVKQGIEQKPVEQRIHFSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQ AQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDYVT HHPGLTPEQTSAGAQAADILSLFCPDADKSCVASNNDQANINIESRSGRSYLPENRAVITPQGVTNW TYQELEATHQALTREGYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGY ARIKEGTGEYGLPTRAERDARGVMLRVYIPRASLERFYRTNTPLENAEEHITQVIGHSLPLRNEAFT GPESAGGEDETVIGWDMAIHAVAIPSTIPGNAYEELAIDEEAVAKEQSISTKPPYKERKDELK 75 Synthetic gene ATGGAAGATGAGCTGAATATTTTTGACGAGTGCCGTAGCCCGTGTTCTCTGACCCCAGAACCTGGCA encoding VCE AACCGATCCAGAGTAAACTGTCAATTCCATCCGATGTGGTTCTGGACGAAGGTGTCCTGTATTACTC GATGACGATCAACGATGAACAAAATGACATTAAAGATGAGGATAAAGGGGAAAGCATCATTACTATC GGAGAGTTCGCGACAGTACGCGCCACCCGTCATTATGTGAACCAGGACGCACCTTTTGGCGTTATTC ACCTGGATATCACGACTGAAAATGGTACAAAAACCTACTCTTATAACCGCAAAGAAGGGGAGTTCGC TATTAATTGGCTGGTCCCGATCGGAGAGGACAGTCCGGCGTCAATTAAAATCTCCGTAGATGAGCTG GACCAACAGCGTAACATTATCGAAGTGCCAAAACTGTACTCGATTGATCTGGATAATCAGACGCTGG AACAATGGAAAACCCAGGGCAACGTTAGCTTTTCTGTCACTCGCCCTGAGCATAATATTGCCATCAG TTGGCCGTCAGTGTCCTATAAAGCAGCTCAAAAAGAAGGTTCGCGTCACAAACGCTGGGCGCATTGG CACACAGGCCTGGCCCTGTGCTGGCTGGTACCGATGGACGCAATTTACAACTATATCACGCAGCAGA ATTGTACCCTGGGTGATAACTGGTTCGGGGGAAGCTATGAGACTGTTGCTGGCACACCAAAAGTGAT TACCGTCAAACAAGGTATCGAACAGAAACCTGTTGAACAACGTATTCATTTTGCTAGCAAAGGCAAT GCCATGAGTGCACTGGCTGCGCACCGCGTATGCGGTGTGCCGCTGGAGACACTGGCCCGTTCACGCA AACCACGTGACCTGACCGATGACCTGAGCTGCGCGTATCAGGCCCAAAATATTGTGTCTCTGTTTGT TGCAACGCGTATCCTGTTCAGTCATCTGGATTCAGTCTTTACTCTGAACCTGGACGAACAGGAGCCG GAAGTAGCTGAGCGCCTGTCCGATCTGCGTCGCATTAATGAAAACAATCCAGGCATGGTGACACAAG TTCTGACCGTCGCGCGTCAGATCTACAACGACTATGTAACGCACCATCCTGGTCTGACTCCGGAACA GACATCGGCCGGGGCACAAGCTGCGGATATTCTGAGCCTGTTCTGTCCAGATGCCGACAAATCTTGC GTGGCAAGTAATAACGATCAGGCTAATATCAACATTGAGTCACGCTCCGGACGTTCGTACCTGCCTG AAAATCGCGCGGTTATCACCCCGCAAGGCGTCACGAACTGGACCTATCAGGAGCTGGAAGCCACTCA CCAGGCACTGACACGTGAAGGTTACGTGTTTGTAGGGTATCATGGAACGAATCACGTTGCTGCGCAA ACCATTGTGAACCGCATCGCCCCGGTCCCACGTGGCAATAACACTGAGAATGAAGAGAAATGGGGTG GCCTGTACGTTGCAACACATGCGGAAGTAGCTCACGGTTATGCCCGCATTAAAGAAGGGACCGGAGA GTATGGCCTGCCTACGCGTGCAGAACGCGACGCGCGTGGTGTGATGCTGCGCGTCTACATCCCGCGT GCTTCGCTGGAGCGCTTCTATCGTACCAACACTCCGCTGGAAAATGCCGAAGAGCATATTACACAGG TTATCGGCCACTCTCTGCCACTGCGCAACGAAGCATTTACGGGTCCTGAAAGTGCGGGGGGAGAGGA TGAAACCGTGATTGGCTGGGACATGGCTATCCATGCCGTAGCAATTCCGTCAACTATTCCAGGTAAT GCGTACGAGGAACTGGCCATCGATGAAGAGGCAGTCGCGAAAGAACAATCCATTTCGACAACCGCCT TATAAAGAGCGTCACCATCATCACCATCACAAAGATGAACTGTAA 76 Protein sequence medelnifdecrspcsltpepgkpiqskisipsdvvldegvlyysmtindeqndikdedkgesiiti corresponding to gefatvratrhyflqdapfgvihldittengtktysynrkegefainwlvpigedspasikisvdel synthetic VCE dqqrniievpkiysidldnqtleqwktqgnvsfsvtrpehniaiswpsvsykaaqkegsrhkrwahw gene htglalcwlvpmdaiynyitqqnctlgdnwfggsyetvagtpkvitvkqgieqkpveqrihfskgna msalaahrvcgvpletlarsrkprdltddlscayqaqnivslfvatrilfshldsvftlnldeqepe vaerlsdlrrinennpgmvtqvltvarqiyndyvthhpgltpeqtsagaqaadilslfcpdadkscv asnndqaniniesrsgrsylpenravitpqgvtnwtyqeleathqaltregyvfvgyhgtnhvaaqt ivnriapvprgnnteneekwgglyvathaevahgyarkegtgeyglptraerdargvmirvyipras lerfyrtntplenaeehitqvighslplrneaftqpesaggedetvigwdmaihavaipstipgnay eelaideeavakegsistkppykerhhhhhhkde 1 77 synthetic gene ATGGGCCCTGAAAATCGCGCGGTTATCACCCCGCAAGGCGTCACGAACTGGACCT encoding ADPRT ATCAGGAGCTGGAAGCCACTCACCAGGCACTGACACGTGAAGGTTACGTGTTTGTAGGGT domain of VCE ATCATGGAACGAATCACGTTGCTGCGCAAACCATTGTGAACCGCATCGCCCCGGTCCCAC GTGGCAATAACACTGAGAATGAAGAGAAATGGGGTGGCCTGTACGTTGCAACACATGCGG AAGTAGCTCACGGTTATGCCCGCATTAAAGAAGGGACCGGAGAGTATGGCCTGCCTACGC GTGCAGAACGCGACGCGCGTGGTGTGATGCTGCGCGTCTACATCCCGCGTGCTTCGCTGG AGCGCTTCTATCGTACCAACACTCCGCTGGAAAATGCCGAAGAGCATATTACACAGGTTA TCGGCCACTCTCTGCCACTGCGCAACGAAGCATTTACGGGTCCTGAAAGTGCGGGGGGAG AGGATGAAACCGTGATTGGCTGGGACATGGCTATCCATGCCGTAGCAATTCCGTCAACTA TTCCAGGTAATGCGTACGAGGAACTGGCCATCGATGAAGAGGCAGTCGCGAAAGAACAAT CCATTTCGACAAAACCGCCTTATAAAGAGCGTCACCATCATCACCATCACAAAGATGAAC TGTAA 78 Protein sequence mgpenravitpqgvtnwtyqeleathqaltregyvfvgyhgtnhvaaqtivnriapvprgnntenee corresponding to lcwgglyvathaevahgyarikegtgeyglptraerdargvmlrvyipraslerfyrtntplenaee ADPRT domain of hitqvighslplrneaftgpesaggedetvigwdmaihavaipstipgnayeeiaideeavakeqsi VCE stkppykerhhhhhhkdel 79 N-GFD-VCE ATGGGCTCCAACGAACTGCATCAGGTGCCGAGCAACTGCGATTGTCTGAACGGCGGTACCTGCGTTT Synthetic gene CCAACAAATATTTTTCTAACATTCACTGGTGTAACTGCCCGAAAAAATTCGGTGGACAACATTGTGA encoding N-GFD- AATCGACGGCGGTGGTGGTTCGGGCGGTGGCGGTTCGGGTGGCGGTGGCAGCTCTAGCAAAGGCAAT VCE with GCCATGAGTGCACTGGCTGCGCACCGCGTATGCGGTGTGCCGCTGGAGACACTGGCCCGTTCACGCA endogenous furin AACCACGTGACCTGACCGATGACCTGAGCTGCGCGTATCAGGCCCAAAATATTGTGTCTCTGTTTGT cleavage site TGCAACGCGTATCCTGTTCAGTCATCTGGATTCAGTCTTTACTCTGAACCTGGACGAACAGGAGCCG GAAGTAGCTGAGCGCCTGTCCGATCTGCGTCGCATTAATGAAAACAATCCAGGCATGGTGACACAAG TTCTGACCGTCGCGCGTCAGATCTACAACGACTATGTAACGCACCATCCTGGTCTGACTCCGGAACA GACATCGGCCGGGGCACAAGCTGCGGATATTCTGAGCCTGTTCTGTCCAGATGCCGACAAATCTTGC GTGGCAAGTAATAACGATCAGGCTAATATCAACATTGAGTCACGCTCCGGACGTTCGTACCTGCCTG AAAATCGCGCGGTTATCACCCCGCAAGGCGTCACGAACTGGACCTATCAGGAGCTGGAAGCCACTCA CCAGGCACTGACACGTGAAGGTTACGTGTTTGTAGGGTATCATGGAACGAATCACGTTGCTGCGCAA ACCATTGTGAACCGCATCCCCCCGGTCCCACGTGGCAATAACACTGAGAATGAAGAGAAATGGGGTG GCCTGTACGTTGCAACACATGCGGAAGTAGCTCACGGTTATGCCCGCATTAAAGAAGGGACCGGAGA GTATGGCCTGCCTACGCGTGCAGAACGCGACGCGCGTGGTGTGATGCTGCGCGTCTACATCCCGCGT GCTTCGCTGGAGCGCTTCTATCGTACCAACACTCCGCTGGAAAATGCCGAAGAGCATATTACACAGG TTATCGGCCACTCTCTGCCACTGCGCAACGAAGCATTTACGGGTCCTGAAAGTGCGGGGGGAGAGGA TGAAACCGTGATTGGCTGGGACATGGCTATCCATGCCGTAGCAATTCCGTCAACTATTCCAGGTAAT GCGTACGAGGAACTGGCCATCGATGAAGAGGCAGTCGCGAAAGAACAATCCATTTCGACAAACCGCC TTATAAGAGCGTCACCATCATCACCATCACAAAGATGAACTGTAAGCGGCCGC 80 Protein sequence MSNELHQVPSN CDCLNGGTCV SNKYFSNIHW CNCPKKFGGQ HCEID corresponding to GGGGSGGGGSGGGGSSSKGNAMSALAAHRVCGVPLETLARSRKPRDLTDDLSCAYQAQNIVSLFVAT synthetic N-GFD- RILFSHLDSVFTLNLDEQEPEVAERLSDLRRINENNPGMVTQVLTVARQIYNDVTHHPGLTPEQTSA VCE with GAQAADILSLFCPDADKSCVASNNDQANINIESRSGRSYLPENRAVITPQGVTNWTYQELEATHQAL endogenous furin TREGYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTGEYGL cleavage site PTRAERDARGVMLRVYIPRASLERFYRTNTPLENAEEEHITQVIGHSLPLRNEAFTGPESAGGEDET VIGWDMAIRAVAIPSTIPGNAYEELAIDEEAVAKEQSISTKPPYKERHHHHHHKDEL 81 Protein sequence Several MSNELHQVPSNCDCLNGGTCVSNKYFSNIHWCNCPKKFGGQHCEIDGGGGSGGGGSGGGGSSSKGNA corresponding to sequences MSALAAHRVCGVPLETLARSIEPDDLTDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPE synthetic N-GPD- in place of VAERLSDLRRINENNPGMVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCV VCE with a underlined ASNNDQANINIESRSGRSYLPENRAVITPQGVTNWTYQELEATHQALTREGYVFVGYHGTNHVAAQT granzyme B region have IVNRIAPVPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTGEYGLPTRAERDARGVMLRYIPRAS cleavage site been tested, LERFYRTNTPLENAEEHITQVIGHSLPLRNEAFTGPESAGGEDETVIGWDMAIHAVAIPSTIPGNAY including EELAIDEEAVAKEQSISTKPPYKERHHHHHHKDEL IEPDSG and IAPDDL. 82 Anti-CD5-VCE ATGgccaacatccagctggtgcagtctggtcctgagctgaagaagcctggtgagactgtcaaaatct synthetic gene cctgcaaggcttctgggtataccttcactaactatgqtatqaactgggtgaagcaggctcctggtaa encoding anti- gggtctgcgttggatgggctggattaacacccacactggtgagcctacttatgctgatgacttcaag CD5-VCE with ggacgttttgccttctctctggaaacttctgccagcactgcctatctccagatcaacaacctcaaaa endogenous furin atgaggacactgctacttacttctgtacacgtcgtggttacgactggtacttcgatgtctggggtgc cleavage site tgggaccacggtgaccgtgttctccgggggaqgtggcagcgggggaggtggcagcggcggcgggagc tccgacatcaagatgacccagtctccttcttccatgtatgcttctctgggtgagcgtgtcactatca cttgcaaggccagccaqgacattaatagctatctgagctggttccatcataaacctgggaaatctcc taagaccctgatctatcgtgctaaccgtctggttgatggggtcccttctcgtttcagcggctctggt tctgggcaagattattctctcaccatcagcagcctggactatgaagatatgggtatttattattgtc aacagtatgatgagtctccttggactttcggtggtggcaccaagctggagatgaaaggctctggcGC TAGCAAAGGCAATGCCATGAGTGCACTGGCTGCGCACCGCGTATGCGGTGTGCCGCTGGAGACACTG GCCCGTTCACGCAAACCACGTGACCTGACCGATGACCTGAGCTGCGCGTATCAGGCCCAAAATATTG TGTCTCTGTTTGTTGCAACGCGTATCCTGTTCAGTCATCTGGATTCAGTCTTTACTCTGAACCTGGA CGAACAGGAGCCGGAAGTAGCTGAGCGCCTGTCCGATCTGCGTCGCATTAATGAAAACAATCCAGGC ATGGTGACACAAGTTCTGACCGTCGCGCGTCAGATCTACAACGACTATGTAACGCACCATCCTGGTC TGACTCCGGAACAGACATCGGCCGGGGCACAAGCTGCGGATATTCTGAGCCTGTTCTGTCCAGATGC CGACAAATCTTGCGTGGCAAGTAATAACGATCAGGCTAATATCAACATTGAGTCACGCTCCGGACGT TCGTACCTGCCTGAAAATCGCGCGGTTATCACCCCGCAAGGCGTCACGACTGGAACCTATCAGGAGC TGGAAGCCACTCACCAGGCACTGACACGTGAAGGTTACGTGTTTGTAGGGTATCATGGAACGAATCA CGTTGCTGCGCAAACCATTGTGAACCGCATCGCCCCGGTCCCACGTGGCAATAACACTGAGAATGAA GAGAAATGGGGTGGCCTGTACGTTGCAACACATGCGGAAGTAGCTCACGGTTATGCCCGCATTAAAG AAGGGACCGGAGAGTATGGCCTGCCTACGCGTGCAGAACGCGACGCGCGTGGTGTGATGCTGCGCGT CTACATCCCGCGTGCTTCGCTGGAGCGCTTCTATCGTACCAACACTCCGCTGGAAAATGCCGAAGAG CATATTACACAGGTTATCGGCCACTCTCTGCCACTGCGCAACGAAGCATTTACGGGTCCTGAAAGTG CGGGGGGAGAGGATGAAACCGTGATTGGCTGGGACATGGCTATCCATGCCGTAGCAATTCCGTCAAC TATTCCAGGTAATGCGTACGAGGAACTGGCCATCGATGAAGAGGCAGTCGCGAAAGAACAATCCATT TCGACAAAACCGCCTTATAAAGAGCGTCACCATCATCACCATCACAAAGATGAACTGTAA 83 Protein sequence Proteins MANIQLVQSGPELKKPGETVKISCKASGYTFTNYGMNWVKQAPGKGLRWMGWINTHTGEPTYADDFK of anti-CD5-VCE with altered GRFAFSLETSASTAYLQINNLKNEDTATYFCTRRGYDWYFDVWGAGTTVTVFSGGGGSGGGGSGGGG with a 15 amino underlined GSSDIKMTQSPSSNYASLGERVTITCKASQDINSYLSWFHHKPGKSPKTLIYRNRLVDGVPSRFSGS acid linker sequence, GSGQDYSLTISSLDYEDMGIYYCQQYDESPWTFGGGTKLEMKGSGASKGNAMSALAAHRVCGVPLET including LARS RKPRDL TDDLSCAYQAQNIVSLFVATRILFSHLDSVFTLNLDEQEPEVAERLSDLRRINENNP IEPDDL, GMVTQVLTVARQIYNDYVTHRPGLTPEQTSAGAQAADILSLFCPDADKSCVASNNDQANINIESRSG IEPDSG, RSYLPENRAVITPQGVTNWTYQELEATHQALTREGYVFVGYHGTNHVAAQTIVNRIAPVPRGNNTEN IAPDDL, EEKWGGLYVATHAEVAHGYARIFCEGTGEYGLPTRAERDARGVMLRVYIPRASLERFYRTNTPLENA IAPDSG, EEHITQVIGHSLPLRNEAFTGPESAGGEDETVIGWDMAIHAVAIPSTIPGNAYEELAIDEEAVAKEQ RVRRAS, SISTKPPYKERHHHHHHKDEL ENLYFQG were also made. 84 Anti-CD19-VCE ATGGCCCAGGTGCAGCTGCAGCAGTCCGGCGCTGAGCTGGTGCGCCCTGGCTCCTCCGTGAAAATCT with a 18 amino CCTGCAAGGCTTCCGGCTACGCTTTCTCCTCCTACTGGATGAACTGGGTGAAGCAGCGCCCTGGCCA acid linker GGGCCTGGAGTGGATCGGCCAAATCTGGCCGGGCGACGGCGACACCAACTACAACGGCAAGTTCAAG GGCAAGGCTACCCTGACCGCTGACGAGTCCTCCTCCACCGCTTACATGCAGCTGTCCTCCCTGGCTT CCGAGGACTCCGCTGTGTACTTGTGCGCTCGCCGCGAGACCACCACCGTGGGCCGCTACTACTACGC TATGGACTACTGGGGCCAGGGCACCTCGGTGACCGTGTCCTCCGGCGGCGGCGGCTCCGGCGGCGGC GGCTCCGGCGGCGGGTCCGGGAGCTCCGACATCCTGCTGACCCAGACCCCGGCTTCCCTGGCTGTGT CCCTGGGCCAGCGCGCTACCATCTCCTGCAAGGCTTCCCAGTCCGTGGACTACGACGGCGACTCCTA CCTGAACTGGTACCAGCAGATCCCGGGCCAGCCGCCGAAGCTGCTGATCTACGACGCTTCCAACCTG GTGTCCGGCATCCCGCCGCGCTTCTCCGGCTCCGGCTCCGGCACCGACTTCACCCTGAACATCCACC CGGTGGAGAAGGTGGACGCTGCTACCTACCACTGCCAGCAGTCCACCGAGGACCCGTGGACCTTCGG CGGCGGCACCAAGCTGGAGATCAAGCGCGGCTCTGGCGCTAGCAAAGGCAATGCCATGAGTGCACTG GCTGCGCACCGCGTATGCGGTGTGCCGCTGGAGACACTGGCCCGTTCACGCAAACCACGTGACCTGA CCGATGACCTGAGCTGCGCGTATCAGGCCCAAAATATTGTGTCTCTGTTTGTTGCAACGCGTATCCT GTTCAGTCATCTGGATTCAGTCTTTACTCTGAACCTGGACGAACAGGAGCCGGAAGTAGCTGAGCGC CTGTCCGATCTGCGTCGCATTAATGAAAACAATCCAGGCATGGTGACACAAGTTCTGACCGTCGCGC GTCAGATCTACAACGACTATGTAACGCACCATCCTGGTCTGACTCCGGAACAGACATCGGCCGGGGC ACAAGCTGCGGATATTCTGAGCCTGTTCTGTCCAGATGCCGACAAATCTTGCGTGGCAAGTAATAAC GATCAGGCTAATATCAACATTGAGTCACGCTCCGGACGTTCGTACCTGCCTGAAAATCGCGCGGTTA TCACCCCGCAAGGCGTCACGAACTGGACCTATCAGGAGCTGGAAGCCACTCACCAGGCACTGACACG TGAAGGTTACGTGTTTGTAGGGTATCATGGAACGAATCACGTTGCTGCGCAAACCATTGTGAACCGC ATCGCCCCGGTCCCACGTGGCAATAACACTGAGAATGAAGAGAAATGGGGTGGCCTGTACGTTGCAA CACATGCGGAAGTAGCTCACGGTTATGCCCGCATTAAAGAAGGGACCGGAGAGTATGGCCTGCCTAC GCGTGCAGAACGCGACGCGCGTGCTGTGATGCTGCGCGTCTACATCCCGCGTGCTTCGCTGGAGCGC TTCTATCGTACCAACACTCCGCTGGAAAATGCCGAAGAGCATATTACACAGGTTATCGGCCACTCTC TGCCACTGCGCAACGAAGCATTTACGGGTCCTGAAAGTGCGGGGGGAGAGGATGAAACCGTGATTGG CTGGGACATGGCTATCCATGCCGTAGCAATTCCGTCAACTATTCCAGGTAATGCGTACGAGGAACTG GCCATCGATGAAGAGGCAGTCGCGAAAGAACAATCCATTTCGACAAAACCGCCTTATAAAGAGCGTC ACCATCATCACCATCACAAAGATGAACTGTAA 85 Anti-CD19-VCE Proteins MAQVQLQQSGAELVRPGSSVKISCKASGYAESSYWNNWKQRPGQGLEWIGQIWPGDGDTNYNGKFKG protein sequence with altered KATLTADESSSTAYMQLSSLASEDSAVYFCARRETTTVGRYYYAMDYWGQGTSVTVSSGGGGSGGGG underlined SGGGSGSSDILLTQTPASLAVSLGQRATISCKASQSVDYDGDSYLNWYQQIPGQPPKLLIYDASNLV sequence, SGIPPRFSGSGSGTDFTLNIHPVEKVDAATYHCQQSTEDPWTFGGGTKLEIKRGSGASKGNAMSAIA including AHRVCGVPLETLARS RKPRDL TDDLSCAYQAQNIVSLFVATRILESHLDSVFTLNLDEQEPEVAERL IEPDDL, SDLRRINENNPGNVTQVLTVARQIYNDYVTHHPGLTPEQTSAGAQAADILSLFCPDADKSCVASNND IEPDSG, QANINIESRSGRSYLPENRAVITPQGVTNWTYQELEATRQALTREGYVFVGYHGTNHVAAQTIVNRI IAPDDL, APVPRGNNTENEEKWGGLYVATHAEVAHGYARIKEGTGEYGLPTRAERDARGVMLRVYIPRASLERF IAPDSG, YRTNTPLENAEEHITQVIGHSLPLRNEAETGPESAGGEDETVIGWDMAIHAVAIPSTIPGNAYEELA RVRRAS, IDEEAVAKEQSISTKPPYKERHHHHRHKDEL ENLYFQG were also made. 86 Synthetic gene ATGGACTACAAGGACGACGACGACAAGcGCATcgccaacatccagctggtgcagtctggtcctgagc encoding anti- tgaagaagcctggtgagactgtcaaaatctcctgcaaggcttctgggtataccttcactaactatgg CD5-PE tatgaactgggtgaagcaggctcctggtaagggtctgcgttggatgggctggattaacacccacact ggtgagcctacttatgctgatgacttcaagggacgttttgccttctctctggaaacttctgccagca ctgcctatctccagatcaacaacctcaaaaatgaggacactgctacttacttctgtacacgtcgtgg ttacgactggtacttcgatgtctggggtgctgggaccacggtgaccgtgttctccgggggaggtggc agcgggggaggtggcagcggcggcgggagctccgacatcaagatgacccagtctccttcttccatgt atgcttctctgggtgagcgtgtcactatcacttgcaaggccagccaggacattaatagctatctgag ctggttccatcataaacctgggaaatctcctaagaccctgatctatcgtgctaaccgtctggttgat ggggtcccttctcgtttcagcggctctggttctgggcaagattattctctcaccatcagcagcctgg actatgaagatatgggtatttattattgtcaacagtatgatgagtctccttggactttcggtggtgg caccaagctggagatgaaaggaggcggaggctccggaggaggaggcgggtccgctagcctGATCGCC CTGACCGCCCACCAGGCCTGCCACCTGCCGCTGGAGACCTTCACCGCTAGCATCGAGCCGGACGGCT GGGAGCAGCTGGAGCAGTGCGGCTACCCGGTGCAGCGCCTGGTGGCCCTGTACCTGGCCGCCCGCCT GTCCTGGAACCAGGTGGACCAGGTGATCCGCAACGCCCTGGCCTCCCCGGCCTCCGGCGGCGACCTG GGCGAGGCCATCCGCGAGCAGCCGGAGCAGGCCCGCCTGGCCCTGACCCTGGCCGCCGCCGAGTCCG AGCGCTTCGTGCGCCAGGGCACCGGCAACGACGAGGCCGGCGCCGCCAACGCCGACGTGGTGTCCCT GACCTGCCCGGTGGCCGCCGGCGAGTGCGCCGGCCCGGCCGACTCCGGCGACGCCCTGCTGGAGCGC AACTACCCGACCGGCGCCGAGTTCCTGGGCGACGGCGGCGACGTGTCCTTCTCCACCCGCGGCACCC AGACCTGGACCGTGGAGCGCCTGCTGCAGGCCCACCGCCAGCTGGAGGAGCGCGGCTACGTGTTCGT GGGCTACCACGGCACCTTCCTGGAGGCCGCCCAGTCCATCGTGTTCGGCGGCGTGCGCGCCCGCTCC CAGGACCTGGACGCCATCTGGCGCGGCTTCTACATCGCCGGCGACCCGGCCCTGGCCTACGGCTACG CCCAGGACCAGGAGCCGGACGCCCGCGGTCGCATCCGCAACGGCGCCCTGCTGCGCGTGTACGTGCC GCGCTCCTCCCTGCCGGGCTTCTACCGCACCTCCCTGACCCTGGCCGCCCCGGAGGCCGCCGGCGAG GTGGAGCGCCTGATCCGCCACCCGCTGCCGCTGCGCCTGGACGCCATCACCGGCCCGGAGGAGGAGG GCGGTCGCCTGGAGACCATCCTGGGCTGGCCGCTGGCCGAGCGCACCGTGGTGATCCCGTCCGCCAT CCCGACCGACCCGCGCAACGTGGGCGGCGACCTGGACCCGTCCTCCATCCCGGACAAGGAGCAGGCC ATCTCCGCCCTGCCGGACTACGCCTCTCAGCCGGGCAAGCCGCCGCACCACCACCACCACCACAAGG ACGAGCTGTAG 87 Anti-CD5-PE MDYKDDDDKGMANIQLVQSGPELKKPGETVKISCKASGYTFTNYGMNWVKQAPGKGLRWMGWINTHT protein sequence GEPTYADDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCTRRGYDWYFDVWGAGTTVTVFSGGGG SGGGGSGGGSSDIKMTQSPSSMYASLGERVTITCKASQDINSYLSWFHHKPGKSPKTLIYRANRLVD GVPSRFSGSGSGQDYSLTISSLDYEDMGIYYCQQYDESPWTFGGGTKLEMKGGGGSGGGGGSASLIA LTAHQACHLPLETFTASIEPDGWEQLEQCGYPVQRLVALYLAARLSWNQVDQVIRNALASPGSGGDL GEMREQPEQARLALTLAAAESERFVRQGTGNDEAGAANADVVSLTCPVAAGECAGPADSGDALLERN YPTGAEFLGDGGDVSFSTRGTQTWTVERLLQAHRQLEERGYVFVGYHGTFLEAAQSIVFGGVRARSQ DLDAIWRGFYIAGDPALAYGYAQDQEPDARGRIRNGALLRVYVPRSSLPGEYRTSLTLAAPEAAGEV ERLIGHPLPLRLDAITGPEEEGGRLETILGWPLAERTVVIPSAIPTDPRNVGGDLDPSSIPDKEQAI SALPDYASQPGKPPHHHHHHKDEL 88 Synthetic gene ATGGGCGTGAAGGTGCTGTTCGCCCTGATCTGCATCGCCGTGGCGctcgccgacaactcgagctaca encoding GrB- aggacgacgacgacaagATCATCGGGGGACATGAGGCCAAGCCCCACTCCCGCCCCTACATGGCTTA anti-CD19 TCTTATGATCTGGGATCAGAAGTCTCTGAAGAGGTGCGGTGGCTTCCTGATACAAGACGACTTCGTG CTGACAGCTGCTCACTGTTGGGGAAGCTCCATAAATGTCACCTTGGGGGCCCACAATATCAAAGAAC AGGAGCCGACCCAGCAGTTTATCCCTGTGAAAAGACCCATCCCCCATCCAGCCTATAATCCTAAGAA CTTCTCCAACGACATCATGCTACTGCAGCTGGAGAGAAAGGCCAAGCGGACCAGAGCTGTTCAGCCC CTCAGGCTACCTAGCAACAAGGCCCAGGTGAAGCCAGGGCAGACATGCAGTGTGGCCGGCTGGGGGC AGACGGCCCCCCTGGGAAAACACTCACACACACTACAAGAGGTGAAGATGACAGTGCAGGAAGATCG AAAGTGCGAATCTGACTTACGCCATTATTACGACAGTACCATTGAGTTGTGCGTGGGGGACCCAGAG ATTAAAAAGACTTCCTTTAAGGGGGACTCTGGAGGCCCTCTTGTGTGTAACAAGGTGGCCCAGGGCA TTGTCTCCTATGGACGAAACAATGGCATGCCTCCACGAGCCTGCACCAAAGTCTCAGCTTTGTACAC TGGATAAAGTAAAACCATGAAACGCTACGCCATGGGAGGCGGAGGCTCCGGAGGAGGAGGGTCCGGG GGCGGCGGAAGCATGGCCCAGGTGCAGCTGCAGCAGTCCGGCGCTGAGCTGGTGCGCCCTGGCTCCT CCGTGAAAATCTCCTGCAAGGCTTCCGGCTACGCTTTCTCCTCCTACTGGATGAACTGGGTGAAGCA GCGCCCTGGCCAGGGCCTGGAGTGGATCGGCCAAATCTGGCCGGGCGACGGCGACACCAACTACAAC GGCAAGTTCAAGGGCAAGGCTACCCTGACCGCTGACGAGTCCTCCTCCACCGCTTACATGCAGCTGT CCTCCCTGGCTTCCGAGGACTCCGCTGTGTACTTCTGCGCTCGCCGCGAGACCACCACCGTGGGCCG CTACTACTACGCTATGGACTACTGGCGCCAGGGCACCTCGGTGACCGTGTCCTCCGGCGGCGGCGGC TCCGGCGGCGGCGGCTCCGGCGGCGGGAGCTCCGACATCCTGCTGACCCAGACCCCGGCTTCCCTGG CTGTGTCCCTGGGCCAGCGCGCTACCATCTCCTGCAAGGCTTCCCAGTCCGTGGACTACGACGGCGA GTCCTACCTGAACTGGTACCAGCAGATCCCGGGCCAGCCGCCGAAGCTGCTGATCTACGACGCTTCC AACCTGGTGTCCGGCATCCCGCCGCGCTTCTCCGGCTCCGGCTCCGGCACCGACTTCACCCTGAACA TCCACCCGGTGGAGAAGGTGGACGCTGCTACCTACCACTGCCAGCAGTCCACCGAGGACCCGTGGAC CTTCGGCGGCGGCACCAAGCTGGAGATCAAGCGCGGTGGTGACATGCATCACCATCACCATCACTGA 89 GrB-anti-CD19 MGVKVLFALICIAVALADNSSYKDDDDKIIGGHEAKPHSRPYMAYLMIWDQKSLKRCGGFLIQDDFV Protein sequence LTAAHCWGSSINVTLGAHNIKEQEPTQQFIPVKRPIPHPAYNPKNFSNDIMLLQLERKAKRTRAVQP LRLPSNKAQVKPGQTCSVAGWGQTAPLGKHSHTLQEVKMTVQEDRKCESDLRHYYDSTIELCVGDPE IKKTSFKGDSGGPLVCNKVAAGIVSYGRNNGMPPRACTKVSSFVHWIKKTMKRYAMGGGGSGGGGSG GGGSMAQVQLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIWPGDGDTNYN GKFKGKATLTADESSSTAYMQLSSLASEDSAVYFCARRETTTVGRYYYAMDYWGQGTSVTVSSGGGG SGGGGSGGGSSDILLTQTPASLAVSLGQRATISCKASQSVDYDGDSYLNWYQQIPGQPPKLLIYDAS NLVSGIPPRFSGSGSGTDFTLNIHPVEKVDAATYHCQQSTEDPWTFGGGTKLEIKRGGDMHHHHHH 90 Synthetic DNA ATGGGTGCCGACGACGTGGTGGACTCCTCCAAGTCCTTCGTGATGGAAAACTTCGCTTCCTACCACG encoding DT- GTACCAAGCCTGGTTACGTGGATTCCATCCAGAAGGGTATCCAGAAGCCTAAGTCCGGTACCCAGGG anit-CD5 TAACTACGACGATGATTGGAAGGGTTTTTACTCCACCGACAACAAGTACGACGCCGCCGGTTACTCC GTGGATAACGAAAACCCTCTGTCCGGTAAGGCCGGTGGTGTGGTGAAAGTGACCTACCCTGGTCTGA CCAAGGTGCTGGCCCTGAAGGTGGATAACGCCGAAACCATCAAGAAGGAGCTGGGTCTGTCCCTGAC CGAACCTCTGATGGAGCAGGTGGGTACCGAAGAGTTTATCAAGAGATTCGGTGATGGTGCCTCCAGA GTGGTGCTGTCCCTGCCTTTCGCCGAGGGTTCCTCCTCCGTGGAATACATCAACAACTGGGAACAGG CCAAGGCCCTGTCCGTGGAACTGGAGATCAACTTTGAAACCAGAGGTAAGAGAGGTCAGGATGCCAT GTACGAGTACatggcccaggcctgtgccggCAACATCGAGCCTGACACCGgttcctccctgtccTGC ATCAACCTGGACTGGGACGTGATCAGAGACAAGACCAAGACCAAGATCGAGTCCCTGAAGGAGCACG GTCCTATCAAGAACAAGATGTCCGAGTCCCCTGCCAAGACCGTGTCCGAGGAGAAGGCCAAGCAGTA CCTGGAGGAGTTCCACCAGACCGCCCTGGAGCACCCTGAGCTGTCCGAGCTGAAGACCGTGACTGGT ACCAACCCTGTGTTCGCCGGTGCCAACTACGCCGCCTGGGCCGTGAACGTGGCCCAGGTGATCGACT CCGAGACCGCCGACAACCTGGAGAAGACCACCGCCGCCCTGTCCATCCTGCCTGGTATCGGTTCCGT GATGGGTATCGCCGACGGTGCCGTGCACCACAACACCGAGGAGATCGTGGCCCAGTCCATCGCCCTG TCCTCCCTGATGGTGGCCCAGGCCATCCCTCTGGTGGGTGAGCTGGTGGACATCGGTTTCGCCGCCT ACAACTTCGTGGAGTCCATCATCAACCTGTTCCAGGTGGTGCACAACTCCTACAACAGACCTGCCTA CTCCCCTGGTCACAAGACCCAGCCTGCCATGGGAGGCGGAGGCTCCGGAGGAGGAGGGTCCGGGGGC GGCGGAAGCATGGCCCAGGTGCAGCTGCAGCAGTCCGGTGCCGAGCTGGTGAGACCTGGTGCCTCCG TGAAGCTGTCCTGCAAGACCTCCGCCTACACCTTCACCAACTACTGGATCAACTGGGTGAAGCAGAG ACCTGGTCAGGGTCTGGAGTGGATCGGTAACATCTACCCTTCCGACTCCTACACCAACTACAACCAG AAGTTCAAGGACAAGGCCACCCTGACCGTGGACAAGTCCTCCTCCACCGCCTACATCCAGCTGTCCT CCCCTACCTCCGAGGACTCCGCCGTGTACTACTGCACCAGAGGTGGTGCCTACTACAGATCCTTCGA CTACTGGGCCCAGGGTACCACGGTGACCGTGTCCTCCGGTGGCGGTGGCTCCGGGGGCGGTGGTTCC GGTGGTGGGAGCTCCGACATCGTGCTGACCCAGTCCCCTGCCATCCTGTCCGCCTCCCCTGGTGAGA AAGTGACCATGACCTGCAGAGCCACCTCCTCCGTGTCCTACATGCACTGGTACCAGCAGAAGCCTGG TTCCTCCCCTAAGCCTTGGATCTACGCCACCTCCAACCTGGCCTCCGGTGTGCCTGCCAGATTCTCC GGTTCCGGTTCCGGTACCTCCTACTCCCTGACCATCTCCAGAGTGGAGGCCGAGGACGCCGCCACCT ACTACTGCCAGCAGTGGTCCTCCAACCCTCCTACCTTCGGTGCCGGTACCATGCTGGAGCTGAAGAG AGGTGGTCACATGCACCATCACCATCATCACTAA 91 Protein sequence MGADDVVDSSKSFVNENFASYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKGFYSTDNKYDAAGYS of DT-anti-CD5 VDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASR VVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNIEPDTGSSLSC INLDWDVIRDKTKTKIESLKEHGPIKNKMSESPAKTVSEEKAKQYLEEFHQTALEHPELSELKTVTG TNPVFAGANYAAWAVNVAQVIDSETADNLEKTTAALSILEGIGSVMGIADGAVHHNTEEIVAQSIAL SSLNVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKTQPAMGGGGSGGGGSGG GGSMAQVQLQQSGAELVRPGASVKLSCKTSAYTFTNYWINWVKQRPGQGLEWIGNIYPSDSYTNYNQ KFKDKATLTVDKSSSTAYIQLSSPTSEDSAVYYCTRGGAYYRSFDYWAQGTTVTVSSGGGGSGGGGS GGGSSDIVLTQSPAILSASPGEKVTMTCRATSSVSYMHWYQQKPGSSPKPWIYATSNLASGVPARFS GSGSGTSYSLTISRVEAEDAATYYCQQWSSNPPTFGAGTMLELKRGGRMHHHHHH 92 pro-aerolysin AEPVYPDQLRLFSLGQGVCGDKYRPVNREEAQSVKSNIVGMNGQWQISGLANGWVIMGPGYNGEIKP Protein Sequence GTASNTWCYPTNPVTGEIPTLSALDIPDGDEVDVQWRLVHDSANFIKFTSYLAHYLGYAWVGGNHSQ YVGEDMDVTRDGDGWVIRGNNDGGCDGYRCGDKTAIKVSNFAYNLDPDSFKHGDVTQSDRQLVKTVV GWAVNDSDTPQSGYDVTLRYDTATNWSKTNTYGLSEKVTTKNKFKWPLVGETELSIEIAANQSWASQ NGGSTTTSLSQSVRPTVPARSKIPVKIELYKADISYPYEFKADVSYDLTLSGFLRWGGNAWYTHPDN RPNWNHTFVIGPYKDKASSIRYQWDKRYIPGEVKWWDWNWTIQQNGLSTMQNNLARVLRPVPAGITG DFSAESQFAGNIEIGAPVPLAADSKVRRARSVDGAGQGLRLEIPLDAQELSGLGFNNVSLSVTPAAN Q 93 GK-aerolysin_(GrB) GKGGSNSAASGEIPTLSALDIPDGDEVDVQWRLVHDSANFIKPTSYLAHYLGYAWVGGNHSQYVGED Protein Sequence MDVTRDGDGWVIRGNNDGGCDGYRCGDKTSIKVSNFAYNLDPDSFKHGDVTQSDRQLVKTVVGWAIN DSDTPQSGYDVTLRYDTATNWSKTNTYGLSEKVTTKNKFKWPLVGETELSIEIAANQSWASQNGGST TTSLSQSVRPTVPAHSKIPVKIELYKADISYPYEFKADVSYDLTLSGFLRWGGNAWYTHPDNRPNWN HTFVIGPYKDKASSIRYQWDKRYIPGEVKWWDWNWTIQQNGLPTMQNNLARVLRPVRAGITGDFSAE SQFAGNIEIGAPVPVAAESKGIEPDSGVEGAGQGLRLEIPLDAQELSGLGFNNVSLSVTPAANQVEH HHHHH 94 GK-aerolysin_(GrB) GGTAAAGGTGGTTCGAATTCTGCAGCTAGCGGAGAAATACCGACTCTGTCTGCCCTGGATATTCCAG DNA Sequence ATGGTGATGAAGTAGATGTGCAATGGCGGCTGGTACATGACAGTGCGAATTTCATCAAACCAACCAG TTATCTGGCCCATTATCTCGGCTATGCCTGGGTAGGGGGGAATCACAGTCAATATGTCGGCGAAGAC ATGGATGTGACCCGTGATGGTGATGGCTGGGTGATCCGTGGCAACAATGACGGTGGCTGCGATGGTT ATCGCTGTGGTGACAAGACCTCCATCAAGGTGAGCAATTTTGCCTACAACCTGGATCCTGACAGTTT CAAGCATGGCGATGTGACCCAGTCCGACCGCCAACTGGTCAAGACGGTGGTGGGGTGGGCTATCAAC GACAGCGACACGCCTCAATCCGGTTATGACGTCACCCTGCGCTACGACACGGCCACCAACTGGTCCA AGACCAACACCTATGGTCTGAGCGAGAAGGTGACCACCAAGAACAAGTTCAAGTGGCCGCTGGTGGG GGAAACCGAGCTCTCCATCGAGATTGCTGCCAACCAGTCCTGGGCCTCCCAGAACGGGGGCTCGACC ACCACCTCTTTGTCCCAGTCCGTGCGCCCGACAGTGCCGGCCCACTCCAAGATCCCGGTGAAGATAG AGCTCTACAAAGCCGACATCTCCTACCCCTACGAGTTCAAGGCCGATGTCAGCTATGACCTGACCCT GAGCGGTTTCCTGCGTTGGGGCGGTAATGCCTGGTATACCCATCCGGACAACCGTCCGAACTGGAAC CACACCTTCGTCATAGGGCCATACAAGGACAAGGCCAGCAGTATCCGCTACCAGTGGGACAAGCGTT ATATCCCGGGTGAAGTGAAGTGGTGGGATTGGAACTGGACCATACAGCAGAACGGTCTGCCTACCAT GCAGAATAACCTGGCCAGGGTGCTGCGCCCGGTGCGGGCCGGGATCACCGGTGATTTCAGTGCCGAG AGCCAGTTTGCCGGCAACATCGAAATCGGCGCTCCCGTGCCGGTCGCTGCCGAATCTAAGGGTATCG AGCCAGATTCTGGTGTTGAAGGTGCCGGTCAGGGTCTGAGACTGGAGATCCCGCTCGATGCACAAGA GCTCTCCGGGCTTGGCTTCAACAATGTCAGCCTCAGCGTGACCCCTGCTGCCAACCAAGTCGAGCAC CACCACCACCACCAC 95 Anti-CD5 LPETG ANIQLVQSGPELKKPGETVKISCKASGYTFTNYGMNWVKQAPGKGLRWMGWINTHTGEPTYADDFKG Protein Sequence RFAFSLETSASTAYLQINNLKNEDTATYFCTRRGYDWYFDVWGAGTTVTVFSGGGGSGGGGSGGGSS DIKMTQSPSSMYASLGERVTITCKASQDINSYLSWFHHKFGKSPKTLIYRANRLVDGVPSRFSGSGS GQDYSLTISSLDYEDMGIYYCQQYDESPWTFGGGTKLEMRLERPHGGGSLPETGGVEHHHHHH 96 Anti-CD5 LPETG GCCAACATCCAGCTGGTGCAGTCTGGTCCTGAGCTGAAGAAGCCTGGTGAGACTGTCAAAATCTCCT DNA Sequence GCAAGGCTTCTGGGTATACCTTCACTAACTATGGTATGAACTGGGTGAAGCAGGCTCCTGGTAAGGG TCTGCGTTGGATGGGCTGGATTAACACCCACACTGGTGAGCCTACTTATGCTGATGACTTCAAGGGA CGTTTTGCCTTCTCTCTGGAAACTTCTGCCAGCACTGCCTATCTCCAGATCAACAACCTCAAAAATG AGGACACTGCTACTTACTTCTGTACACGTCGTGGTTACGACTGGTACTTCGATGTCTGGGGTGCTGG GACCACGGTGACCGTGTTCTCCGGGGGAGGTGGCAGCGGGGGAGGTGGCAGCGGCGGCGGGAGCTCC GACATCAAGATGACCCAGTCTCCTTCTTCCATGTATGCTTCTCTGGGTGAGCGTGTCACTATCACTT GCAAGGCCAGCCAGGACATTAATAGCTATCTGAGCTGGTTCCATCATAAACCTGGGAAATCTCCTAA GACCCTGATCTATCGTGCTAACCGTCTGGTTGATGGGGTCCCTTCTCGTTTCAGCGGCTCTGGTTCT GGGCAAGATTATTCTCTCACCATCAGCAGCCTGGACTATGAAGATATGGGTATTTATTATTGTCAAC AGTATGATGAGTCTCCTTGGACTTTCGGTGGTGGCACCAAGCTGGAGATGCGTCTCGAGCGGCCGCA TGGCGGCGGCTCCCTGCCAGAGACTGGCGGGGTCGAGCACCACCACCACCACCAC 97 SortaseA ANIQLVQSGPELKKPGETVKISCKASGYTFTNYGMNWVKQAPGKGLRWMGWINTHTGEPTYADDFKG conjugated anti- RFAFSLETSASTAYLQINNLKNEDTATYFCTRRGYDWYFDVWGAGTTVTVESGGGGSGGGGSGGGSS CD5-aerolysin_(GrB) DIKMTQSPSSMYASLGERVTITCKASQDINSYLSWFHHKPGKSPKTLIYRANRLVDGVPSRFSGSGS Protein Sequence GQDYSLTISSLDYEDMGIYYCQQYDESPWTFGGGTKLEMRLERPHGGGSLPETGKGGSNSAASGEIP TLSALDIPDGDEVDVQWRLVHDSANFIKPTSYLAHYLGYAWVGGNHSQYVGEDMDVTRDGDGWVIRG NNDGGCDGYRCGDKTSIKVSNFAYNLDPDSFKHGDVTQSDRQLVKTVVGWAINDSDTPQSGYDVTLR YDTATNWSKTNTYGLSEKVTTKNKFKWPLVGETELSIEIAANQSWASQNGGSTTTSLSQSVRPTVPA HSKIPVKIELYKADISYPYEFKADVSYDLTLSGFLRWGGNAWYTHPDNRPNWNHTFVIGPYKDKASS IRYQWDKRYIPGEVKWWDWNWTIQQNGLPTMQNNLARVLRPVRAGITGDFSAESQFAGNIEIGAPVP VAAESKGIEPDSGVEGAGQGLRLEIPLDAQELSGLGFNNVSLSVTPAANQVEHHHHHH 98 Protein Sequence MKYLLPTAAAGLLLLAAQPAMAANSAQVQLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRP for anti-CD19- GQGLEWIGQIWPGDGDTNYNGKFKGKATLTADESSSTAYMQLSSLASEDSAVYFCARRETTTVGRYY LPETG YAMDYWGQGTSVTVSSGGGGSGGGGSGGGGSGSSDILLTQTPASLAVSLGQRATISCKASQSVDYDG (underlined is DSYLNWYQQIPGQPPKLLIYDASNLVSGIPPRFSGSGSGTDFTLNIHPVEKVDAATYHCQQSTEDPW signal sequence) TFGGGTKLEIKRGGLERPHGGGSLPETGGVEHHHHHH 99 DNA Sequence for (underlined ATGAAATACCTGCTGCCGACCGCTGCTGCTGGTCTGCTGCTCCTCGCTGCCCAGCCGGCGATGGCCG anti-CD19-LPETG is signal CGAATTCTGCCCAGGTGCAGCTGCAGCAGTCCGGCGCTGAGCTGGTGCGCCCTGGCTCCTCCGTGAA (underlined is sequence) AATCTCCTGCAAGGCTTCCGGCTACGCTTTCTCCTCCTACTGGATGAACTGGGTGAAGCAGCGCCCT signal sequence) GGCCAGGGCCTGGAGTGGATCGGCCAAATCTGGCCGGGCGACGGCGACACCAACTACAACGGCAAGT TCAAGGGCAAGGCTACCCTGACCGCTGACGAGTCCTCCTCCACCGCTTACATGCAGCTGTCCTCCCT GGCTTCCGAGGACTCCGCTGTGTACTTCTGCGCTCGCCGCGAGACCACCACCGTGGGCCGCTACTAC TACGCTATGGACTACTGGGGCCAGGGCACCTCGGTGACCGTGTCCTCCGGGGGAGGTGGCAGCGGTG GAGGTGGCAGCGGCGGCGGGGGTTCCGGGAGCTCCGACATCCTGCTGACCCAGACCCCGGCTTCCCT GGCTGTGTCCCTGGGCCAGCGCGCTACCATCTCCTGCAAGGCTTCCCAGTCCGTGGACTACGACGGC GACTCCTACCTGAACTGGTACCAGCAGATCCCGGGCCAGCCGCCGAAGCTGCTGATCTACGACGCTT CCAACCTGGTGTCCGGCATCCCGCCGCGCTTCTCCGGCTCCGGCTCCGGCACCGACTTCACCCTGAA CATCCACCCGGTGGAGAAGGTGGACGCTGCTACCTACCACTGCCAGCAGTCCACCGAGGACCCGTGG ACCTTCGGCGGCGGCACCAAGCTGCAGATCAAGCGCGGTGGTCTCGAGCGGCCGCATGGCGGCGGCT CCCTGCCAGAGACTGGCGGGGTCGAGCACCACCACCACCACCAC 100 Protein Sequence ANSAQVQLQQSGELVRPGSSVKISCKSGYAFSSYWMNWVKQRPGQGLEWIGQIWFGDGDTNYNGKFK for anti-CD19- GKATLTADESSSTAYMQLSSLASEDAVYFCARRETTTVGRYYYAMDYWGQGTSVTVSSGGGGSGGGG aerolysin_(GrB) SGGGGSGSSDILLTQTPASLAVSLGQRATISCKASQSVDYDGDSYLNWYQQIPGQPPKLLIYDASNL VSGIPPRFSGSGSGTDFTLNIHPVEKVDAATYHCQQSTEDPWTFGGGTKLEIKRGGLERPHGGGSLP ETGKGGSNSAASGEIPTLSALDIPDGDEVDVQWRLVHDSANFIKPTSYLAHYLGYAWVGGNHSQYVG EDMDVTRDGDGWVIRGWNDGGCDGYRCGDKTSIKVSNFAYNLDPDSFKHGDVTQSDRQLVKTVVGWA INDSDTPQSGYDVTLRYDTATNWSKTNTYGLSEKVTTKNKFKWPLVGETELSIEIAANQSWASQNGG STTTSLSQSVRPTVPAHSKIPVKIELYKADISYPYEFKADVSYDLTLSGFLRWGGNAWYTHPDNRPN WNHTFVIGPYKDKASSIRYQWDKRYIPGEVKWWDWNWTIQQNGLPTMQNNLARVLRPVRAGITGDFS AESQFAGNIEIGAPVPVAAESKGIEPDSGVEGAGQGLRLEIPLDAQELSGLGFNNVSLSVTPAANQV EHHHHHH 101 Protein Sequence MKYLLPTAAAGLLLLAAQPAMAGKGGSNSAASGEIPTLSALDIPDGDEVDVQWRLVHDSANFIKPTS for GK- YLAHYLGYAWVGGNHSQYVGEDMDVTRDGDGWVIRGNNDGGCDGYRCGDKTSIKVSNFAYNLDPDSF aerolysin_(TEV) KHGDVTQSDRQLVKTVVGWAINDSDTPQSGYDVTLRYDTATNWSKTNTYGLSEKVTTKNKFKWPLVG ETELSIEIAANQSWASQNGGSTTTSLSQSVRPTVPAHSKIPVKIELYKADISYPYEFKADVSYDLTL SGFLRWGGNAWYTHPDNRPNWNHTFVIGPYKDKASSIRYQWDKRYIPGEVKWWDWNWTIQQNGLPTM QNNLARVLRPVRAGITGDFSAESQFAGNIEIGAPVPVAAESKENLYFQGVEGAGQGLRLEIPLDAQE LSGLGFNNVSLSVTPAANQVEHHHHHH 102 DNA Sequence for ATGAAATACCTGCTGCCGACCGCTGCTGCTGGTCTGCTGCTCCTCGCTGCCCAGCCGGCGATGGCCG GK-aerolysin_(TEV) GTAAAGGTGGTTCGAATTCTGCAGCTAGCGGAGAAATACCGACTCTGTCTGCCCTGGATATTCCAGA TGGTGATGAAGTAGATGTGCAATGGCGGCTGGTACATGACAGTGCGAATTTCATCAAACCAACCAGT TATCTGGCCCATTATCTCGGCTATGCCTGGGTAGGGGGGAATCACAGTCAATATGTCGGCGAAGACA TGGATGTGACCCGTGATGGTGATGGCTGGGTGATCCGTGGCAACAATGACGGTGGCTGCGATGGTTA TCGCTGTGGTGACAAGACCTCCATCAAGGTGAGCAATTTTGCCTACAACCTGGATCCTGACAGTTTC AAGCATGGCGATGTGACCCAGTCCGACCGCCAACTGGTCAAGACGGTGGTGGGGGTGGCTATCAACG ACAGCGACACGCCTCAATCCGGTTATGACGTCACCCTGCGCTACGACACGGCCACCAACTGGTCCAA GACCAACACCTATGGTCTGAGCGAGAAGGTGACCACCAAGTTCAAGTTCAAGTGGCCGCTGGTGGGG GAAACCGAGCTCTCCATCGAGATTGCTGCCAACCAGTCCTGGGCCTCCCAGAACGGGGGCTCGACCA CCACCTCTTTGTCCCAGTCCGTGCGCCCGACAGTGCCGGCCCACTCCAAGATCCCGGTGAAGATAGA GCTCTACAAAGCCGACATCTCCTACCCCTACGAGTTCAAGGCCGATGTCAGCTATGACCTGACCCTG AGCGGTTTCCTGCGTTGGGGCGGTAATGCCTGGTATACCCATCCGGACAACCGTCCGAACTGGAACC ACACCTTCGTCATAGGGCCATACAAGGACAAGGCCAGCAGTATCCGCTACCAGTGGGACAAGCGTTA TATCCCGGGTGAAGTGAAGTGGTGGGATTGGAACTGGACCATACAGCAGAACGGTCTGCCTACCATG CAGAATAACCTGGCCAGGGTGCTGCGCCCGGTGCGGGCCGGGATCACCGGTGATTTCAGTGCCGAGA GCCAGTTTGCCGGCAACATCGAAATCGGCGCTCCCGTGCCGGTCGCTGCCGAATCTAAGGAGAACCT GTACTTCCAAGGTGTTGAAGGTGCCGGTCAGGGTCTGAGACTGGAGATCCCGCTCGATGCACAAGAG CTCTCCGGGCTTGGCTTCAACAATGTCAGCCTCAGCGTGACCCCTGCTGCCAACCAAGTCGAGCACC ACCACCACCACAC 103 Protein Sequence MKYLLPTAAAGLLLLAAQPAMAGKGGSNSAASGEIPTLSALDIPDGPEVDVQWRLVHDSANFIKPTS for GK- YLAHYLGYAWVGGNHSQYVGEDMDVTRDGDGWVIRGNNDGGCDGYRCGDKTSIKVSNFAYNLDPDSF aerolysin_(GrB) KHGDVTQSDRQLVKTVVGWAINDSDTPQSGYDVTLRYDTATNWSKTNTYGLSEKVTTKNKFKWPLVG ETELSIEIAANQSWASQNGGSTTTSLSQSVRPTVPAHSKIPVKIELYKADISYPYEFKADVSYDLTL SGFLRWGGNAWYTHPDNRPNWNHTFVIGPYKDKASSIRYQWDKRYIPGEVKWWDWNWTIQQNGLPTM QNNLARVLRPVRAGITGDFSAESQFAGNIEIGAPVPVAAESKGIEPDSGVEGAGQGLRLEIPLDAQE LSGLGFNNVSLSVTPAANQVEHHHHHH 104 DNA Sequence for ATGAAATACCTGCTGCCGACCGCTGCTGCTGGTCTGCTGCTCCTCGCTGCCCAGCCGGCGATGGCCG GK-aerolysin_(GrB) GTAAAGGTGGTTCGAATTCTGCAGCTAGCGGAGAAATACCGACTCTGTCTGCCCTGGATATTCCAGA TGGTGATGAAGTAGATGTGCAATGGCGGCTGGTACATGACAGTGCGAATTTCATCAAACCAACCAGT TATCTGGCCCATTATCTCGGCTATGCCTGGGTAGGGGGGAATCACAGTCAATATGTCGGCGAAGACA TGGATGTGACCCGTGATGGTGATGGCTGGGTGATCCGTGGCAACAATGACGGTGGCTGCGATGGTTA TCGCTGTGGTGACAAGACCTCCATCAAGGTGACCAATTTTGCCTACAACCTGGATCCTGACAGTTTC AAGCATGGCGATGTGACCCAGTCCGACCGCCAACTGGTCAAGACGGTGGTGGGGTGGGCTATCAACG ACAGCGACACGCCTCAATCCGGTTATGACGTCACCCTGCGCTACGACACGGCCACCAACTGGTCCAA GACCAACACCTATGGTCTGAGCGAGAAGGTGACCACCAAGAACAAGTTCAAGTGGCCGCTGGTGGGG GAAACCGAGCTCTCCATCGAGATTGCTGCCAACCAGTCCTGGGCCTCCCAGAACGGGGGCTCGACCA CCACCTCTTTGTCCCAGTCCGTGCGCCCGACAGTGCCGGCCCACTCCAAGATCCCGGTGAAGATAGA GCTCTACAAAGCCGACATCTCCTACCCCTACGAGTTCAAGGCCGATGTCAGCTATGACCTGACCCTG AGCGGTTTCCTGCGTTGGGGCGGTAATGCCTGGTATACCCATCCGGACAACCGTCCGAACTGGAACC ACACCTTCGTCATAGGGCCATACAAGGACAAGGCCAGCAGTATCCGCTACCAGTGGGACAAGCGTTA TATCCCGGGTGAAGTGAAGTGGTGGGATTGGAACTGGACCATACAGCAGAACGGTCTGCCTACCATG CAGAATAACCTGGCCAGGGTGCTGCGCCCGGTGCGGGCCGGGATCACCGGTGATTTCAGTGCCGAGA GCCAGTTTGCCGGCAACATCGAAATCGGCGCTCCCGTGCCGGTCGCTGCCGAATCTAAGGGTATCGA GCCAGATTCTGGTGTTGAAGGTGCCGGTCAGGGTCTGAGACTGGAGATCCCGCTCGATGCACAAGAG CTCTCCGGGCTTGGCTTCAACAATGTCAGCCTCAGCGTGACCCCTGCTGCCAACCAAGTCGAGCACC ACCACCACCACCAC 105- Protein Sequence MANIQLVQSGPELKKPGETVKISCKASGYTFTNYGMNWVKQAPGKGLRWMGWINTHTGEPTYADDFK for anti-CD5- GRFAFSLETSASTAYLQINNLKNEDTATYFCTRRGYDWYFDVWGAGTTVTVFSCCGGSGGGGSGGGS LPETQ SDIKMTQSPSSMYASLGERVTITCKASQDINSYLSWFHHKPGKSPKTLIYRANRLVDGVPSRFSGSG SGQDYSLTISSLDYEDMGIYYCQQYDESPWTFGGGTKLEMRLERPHGGGSLPETGGVEHHHHHH 106 DNA Sequence for ATGGCCAACATCCAGCTGGTGCAGTCTGGTCCTGAGCTGAAGAAGCCTGGTGAGACTGTCAAAATCT anti-CD5-LPETG CCTGCAAGGCTTCTGGGTATACCTTCACTAACTATGGTATGAACTGGGTGAAGCAGGCTCCTGGTAA GGGTCTGCGTTGGATGGGCTGGATTAACACCCACACTGGTGAGCCTACTTATGCTGATGACTTCAAG GGACGTTTTGCCTTCTCTCTGGAAACTTCTGCCAGCACTGCCTATCTCCAGATCAACAACCTCAAAA ATGAGGACACTGCTACTTACTTCTGTACACGTCGTGGTTACGACTGGTACTTCGATGTCTGGGGTGC TGGGACCACGGTGACCGTGTTCTCCGGGGGAGGTGGCAGCGGGGGAGGTGGCAGCGGCGGCGGGAGC TCCGACATCAAGATGACCCAGTCTCCTTCTTCCATGTATGCTTCTCTGGGTGAGCGTGTCACTATCA CTTGCAAGGCCAGCCAGGACATTAATAGCTATCTGAGCTGGTTCCATCATAAACCTGGGAAATCTCC TAAGACCCTGATCTATCGTGCTAACCGTCTGGTTGATGGGGTCCCTTCTCGTTTCAGCGGCTCTGGT TCTGGGCAAGATTATTCTCTCACCATCAGCAGCCTGGACTATGAAGATATGGGTATTTATTATTGTC AACAGTATGATGAGTCTCCTTGGACTTTCGGTGGTGGCACCAAGCTGCAGATGCGTCTCGAGCGGCC GCATGGCGGCGGCTCCCTGCCAGAGACTGGCGGGGTCGAGCACCACCACCACCACCAC 107 Protein Sequence         10         20         30         40         50         60 forTrx-DT-CCPE MGSDKIIHLT DDSFDTDVLK ADGAILVDFW AHWCGPCKMI APILDEIADE YQGKLTVAKL         70         80         90        100        110        120 NIDHNPGTAP KYGIRGIPTL LLFKNGEVAA TKVGALSKGQ LKEFLDANLA GSGSCDDDDK        130        140        150        160        170        180 LGIDPFTEML YFQGGADDVV DSSKSEVMEM FASYHGTKPG YVDSIQKGIQ KPKSGTQGNY        190        200        210        220        230        240 DDDWKGFYST DNKYDAAGYS VDNENPLSGK AGGVVKVTYP GLTKVLALKV DNAETIKKEL        250        260        270        280        290        300 GLSLTEPLME QVGTEEFIKR FGDGASRVVL SLPFAEGSSS VEYINNWEQA KALSVELEIN        310        320        330        340        350        360 FETRGKRGQD AMYEYMAQAC AGNIEPDTGS SLSCINLDWD VIRDKTKTKI ESLKEHGPIK        370        380        390        400        410        420 NKMSESPAKT VSEEKAKQYL EEFHQTALEH FELSELKTVT GTNPVFAGAN YAAWAVNVAQ        430        440        450        460        470        480 VIDSETADNL EKTTAALSIL PGIGSVMGIA DGAVHHNTEE IVAQSIALSS LMVAQATPLV        490        500        510        520        530        540 GELVDIGFAA YNFVESIINL FQVVHNSYNR PAYSPGHKTQ PAMGGGGSGG GGSGGGGSKG        550        560        570        580        590        600 ELERCVLTVP STDIEKEILD LAAATERLNL TDALNSNPAG NLYDWRSSNS YPWTQKLNLH        610        620        630        640        650        660 LTITATGQKY RILASKIVDF NIYSNNFNNL VKLEQSLGDG VKDHYVDISL DAGQYVLVMK        670        680        690        700 ANSSYSGNYP YSILFQKFKL EGKPIPNPLL GLDSTRTGHH HHHH 108 DNA Sequence for CC ATG GGATCTGATAAAATTATTCATCTGACTGATGATTCTTTTGATACTGATGTACTTAAGGCAGA Trx-DT-CCPE TGGTGCAATCCTGGTTGATTTCTGGGCACACTGGTGCGGTCCGTGCAAAATGATCGCTCCGATTCTG GATGAAATCGCTGACGAATATCAGGGCAAACTGACCGTTGCAAAACTGAACATCGATCACAACCCGG GCACTGCGCCGAAATATGGCATCCGTGGTATCCCGACTCTGCTGCTGTTCAAAAACGGTGAAGTGGC GGCAACCAAAGTGGGTGCACTGTCTAAAGGTCAGTTGAAAGAGTTCCTCGACGCTAACCTGGCCGGC TCTGGATCCGGTGATGACGATGACAAGCTGGGAATTGATCCCTTCACCGAGAACCTGTACTTCCAGG GCGGTGCCGACGACGTGGTGGACTCCTCCAAGTCCTTCGTGATGGAAAACTTCGCTTCCTACCACGG TACCAAGCCTGGTTACGTGGATTCCATCCAGAAGGGTATCCAGAAGCCTAAGTCCGGTACCCAGGGT AACTACGACGATGATTGGAAGGGTTTTTACTCCACCGACAACAAGTACGACGCCGCCGGTTACTCCG TGGATAACGAAAACCCTCTGTCCGGTAAGGCCGGTGGTGTGGTGAAAGTGACCTACCCTGGTCTGAC CAAGGTGCTGGCCCTGAAGGTGGATAACGCCGAAACCATCAAGAAGGAGCTGGGTCTGTCCCTGACC GAACCTCTGATGGAGCAGGTGGGTACCGAAGAGTTTATCAAGAGATTCGGTGATGGTGCCTCCAGAG TGGTGCTGTCCCTGCCTTTCGCCGAGGGTTCCTCCTCCGTGGAATACATCAACAACTGGGAACAGGC CAAGGCCCTGTCCGTGGAACTGGAGATCAACTTTGAAACCAGAGGTAAGAGAGGTCAGGATGCCATG TACGAGTAcatggcccaggcctgtgccggCAACATCGAGCCTGACACCGgttcctccctgtccTGCA TCAACCTGGACTGGGACGTGATCAGAGACAAGACCAAGACCAAGATCGAGTCCCTGAAGGAGCACGG TCCTATCAAGAACAAGATGTCCGAGTCCCCTGCCAAGACCGTGTCCGAGGAGAAGGCCAAGCAGTAC CTGGAGGAGTTCCACCAGACCGCCCTGGAGCACCCTGAGCTGTCCGAGCTGAAGACCGTGACTGGTA CCAACCCTGTGTTCGCCGGTGCCAACTACGCCGCCTGGGCCGTGAACGTGGCCCAGGTGATCGACTC CGAGACCGCCGACAACCTGGAGAAGACCACCGCCGCCCTGTCCATCCTGCCTGGTATCGGTTCCGTG ATGGGTATCGCCGACGGTGCCGTGCACCACAACACCGAGGAGATCGTGGCCCAGTCCATCGCCCTGT CCTCCCTGATGGTGGCCCAGGCCATCCCTCTGGTGGGTGAGCTGGTGGACATCGGTTTCGCCGCCTA CAACTTCGTGGAGTCCATCATCAACCTGTTCCAGGTGGTGCACAACTCCTACAACAGACCTGCCTAC TCCCCTGGTCACAAGACCCAGCCTGCCATGGGAGGCGGAGGCTCCGGAGGAGGAGGGTCCGGGGGCG GCGGAAGCaagggcgagctcGAAAGATGTGTTTTAACAGTTCCATCTACAGATATAGAAAAAGAAAT CCTTGATTTAGCTGCTGCTACAGAAAGATTAAATTTAACTGATGCATTAAACTCAAATCCAGCTGGT AATTTATATGATTGGCGTTCTTCTAACTCATACCCTTGGACTCAAAAGCTCAATTTACACTTAACAA TTACAGCTACTGGACAAAAATATAGAATCTTAGCTAGCAAAATTGTTGATTTTAATATTTATTCAAA TAATTTTAATAATCTAGTGAAATTAGAACAGTCCTTAGGTGATGGAGTAAAAGATCATTATGTTGAT ATAAGTTTAGATGCTGGACAATATGTTCTTGTAATGAAAGCTAATTCATCATATAGTGGAAATTACC CTTATTCAATATTATTTCAAAAATTTaagcttGAAGGTAAGCCTATCCCTAACCCTCTCCTCGGTCT CGATTCTACGCGTACCGCTCATCATCACCATCACCATTGAgtttaaac 109 Protein Sequence         10         20         30         40         50         60 for DT-CCPE GGADDVVDSS KSFVMENFAS YHGTKPGYVD SIQKGIQKPK SGTQGNYDDD WKGFYSTDNK         70         80         90        100        110        120 YDAAGYSVDN ENPLSGKAGG VVKVTYPGLT KVLALKVDNA ETIKKELGLS LTEPLMEQVG        130        140        150        160        170        180 TEEFIKRFGD GASRVVLSLP FAEGSSSVEY INNWEQAKAL SVELEINFET RGKRGQDAMY        190        200        210        220        230        240 EYMAQACAGN IEPDTGSSLS CINLDWDVIR DKTKTKIESL KEHGPINKM SESPAKTVSE        250        260        270        280        290        300 EKAKQYLEEE HQTALEHPEL SELKTVTGTN PVFAGANYAA WAVNVAQVID SETADNLEKT        310        320        330        340        350        360 TAALSILPGI GSVMGIADGA VHHNTEEIVA QSIALSSLMV AQAIFLVGEL VDICFAAYNF        370        380        390        400        410        420 VESIINLFQV VHNSYNRPAY SPGHKTQPAM GGGGSGGGGS GGGGSKGELE RCVLTVPSTD        430        440        450        460        470        480 IEKEILDLAA ATERLNLTDA LNSNPAGNLY DWRSSNSYPW TQKLNLHLTI TATGQKYRIL        490        500        510        520        530        540 ASKIVDFNIY SNNFNNLVKL EQSLGDCVKD HYVDISLDAG QYVLVMKANS SYSGNYPYSI        550        560        570 LFQKFKLEGK PIPNPLLGLD STRTGHHHHH H 110 Protein Sequence         10         20         30         40         50         60 for Pro-GrB- GQRAGCCAVS SFWQRIARGQ QKLAATMGVK VLFALICIAV ALADNSSYKD DDDKIIGGHE (YSA)₂         70         80         90        100        110        120 (expressed AKPHSRPYMA YLMIWDQKSL KRCGGFLIQD DFVLTAAHCW GSSINVTLGA HNIKEQEPTQ in pEAK15)        130        140        150        160        170        180 QFIPVKRPIP HPAYNPKNFS NDIMLLQLER KAKRTRAVQP LRLFSNKAQV KPGQTCSVAG        190        200        210        220        230        240 WGOTAPLGKH SHTLQEVKMT VQEDRKCESD LRHYYDSTIE LCVGDPEIKK TSFKGDSGGP        250        260        270        280        290        300 LVCNKVAQGI VSYGRNNGNP PRACTKVSSF VHWIKKTMKR YAMGGGGSYS AYPDSVPMMS        310        320        330 GGGGSYSAYP DSVPMMSGGG GSHHHHHH 111 DNA Sequence for GGGCAACGTGCTGGTTGTTGTGCTGTCTCATCATTTTGGCAAAGAATTgcacgaggtcagcagAagc Pro-GrB-(YSA)₂ ttgccgccaccATGGGCGTGAAGGTGCTGTTCGCCCTGATCTGCATCGCCGTGGCGctcgccgacaa ctcgagctacaaggacgacgacgacaagATCATCGGGGGACATGAGGCCAAGCCCCACTCCCGCCCC TACATGGCTTATCTTATGATCTGGGATCAGAAGTCTCTGAAGAGGTGCGGTGGCTTCCTGATACAAG ACGACTTCGTGCTGACAGCTGCTCACTGTTGGGGAAGCTCCATAAATGTCACCTTGGGGGCCCACAA TATCAAAGAACAGGAGCCGACCCAGCAGTTTATCCCTGTGAAAAGACCCATCCCCCATCCAGCCTAT AATCCTAAGAACTTCTCCAACGACATCATGCTACTGCAGCTGGAGAGAAAGGCCAAGCGGACCAGAG CTGTGCAGCCCCTCAGGCTACCTAGCAACAAGGCCCAGGTGAAGCCAGGGCAGACATGCAGTGTGGC CGGCTGGGGGCAGACCGCCCCCCTGGGAAAACACTCACACACACTACAAGAGGTGAAGATCACAGTG CAGGAAGATCGAAAGTGCGAATCTGACTTACGCCATTATTACGACAGTACCATTGAGTTGTGCGTGG GGGACCCAGAGATTAAAAAGACTTCCTTTAAGGGGGACTCTGGAGGCCCTCTTGTGTGTAACAAGGT GGCCCAGGGCATTGTCTCCTATGGACGAAACAATGGCATGCCTCCACGAGCCTGCACCAAAGTCTCA AGCTTTGTACACTGGATAAAGAAAACCATGAAACGCTACGCCATGGGTGGCGGTGGCTCTTACTCCG CTTATCCTGATTCCGTTCCAATGATGTCTGGCGGTGGCGGTTCCTATTCTGCCTACCCAGACTCCGT CCCTATGATGTCTGGTGGCCGTGGCTCCCATCACCATCACCATCACAAGGATTAAAAGCTTGAAGTC CGAGGAATTCGGGACAgcggccgc 112 Protein Sequence         10         20         30         40         50         60 for Activated IIGGREAKPH SRPYMAYLMI WDQKSLKRCG GFLIQDDFVL TAAHCWGSSI NVTLGAHNIK GrB-(YSA)₂         70         80         90        100        110        120 EQEPTQQFIP VKRPIPHPAY NPKNFSNDIM LLQLERKAKR TRAVQPLRLP SNKAQVKPGQ        130        140        150        160        170        180 TCSVAGWGQT APLGKHSHTL QEVKMTVQED RKCESDLRHY YDSTIELCVG DPEIKKTSFK        190        200        210        220        230        240 GDSGGPLVCN KVAQGIVSYG RNNGMPPRAC TKVSSFVHWI KKTMKRYAMG GGGSYSAYPD        250        260        270 SVPMMSGGGG SYSAYPDSVP MMSGGGGSHH HHHH 113 DNA Sequence for ATCATCGGGGGACATGAGGCCAAGCCCCACTCCCGCCCCTACATGGCTTATCTTATGATCTGGGATC GrB-(YSA)₂ AGAAGTCTCTGAAGAGGTGCGGTGGCTTCCTGATACAAGACGACTACGTGCTGACAGCTGCTCACTG TTGGGGAAGCTCCATAAATGTCACCTTGGGGGCCCACAATATCAAAGAACAGGAGCCGACCCAGCAG TTTATCCCTGTGAAAAGACCCATCCCCCATCCAGCCTATAATCCTAAGAACTTCTCCAACGACATCA TGCTACTGCAGCTGGAGAGAAAGGCCAAGCGGACCAGAGCTGTGCAGCCCCTCAGGCTACCTAGCAA CAAGGCCCAGGTGAAGCCAGGGCAGACATGCAGTGTGGCCGGCTGGGGGCAGACGGCCCCCCTGGGA AAACACTCACACACACTACAAGAGGTGAAGATGACAGTGCAGGAAGATCGAAAGTGCGAATCTGACT TACGCCATTATTACGACAGTACCATTGAGTTGTGCGTGGGGGACCCAGAGATTAAAAAGACTTCCTT TAAGGGGGACTCTGGAGGCCCTCTTGTGTGTAACAAGGTGGCCCAGGGCATTGTCTCCTATGGACGA AACAATGGCATGCCTCCACGAGCCTGCACCAAAGTCTCAAGCTTTGTACACTGGATAAAGAAAACCA TGAAACGCTACGCCATGGGTGGCGGTGGCTCTTACTCCGCTTATCCTGATTCCGTTCCAATGATGTC TGGCGGTGGCGGTTCCTATTCTGCCTACCCAGACTCCGTCCCTATGATGTCTGGTGGCGGTGGCTCC CATCACCATCACCATCACAAGGATTAAAAGCTT 114 Protein Sequence Proteins         10         20         30         40         50         60 for Trx-DT^(A)- with MGSDKIIHLT DDSFDTDVLK ADGAILVDFW AHWCGPCKMI APILDEIADE YQGKLTVAKL anti-CD19 different         70         80         90        100        110        120 underlined NIDRNPGTAP KYGIRGIPIL LLFKNGEVAA TKVGALSKGQ LKEFLDANLA GSGSGDDDDK sequence,        130        140        150        160        170        180 including LGIDPFTGAD DVVDSSKSFV MEMFASYHGT KPGYVDSIQK GIQKPKSGTQ GNYDDDWKGF RVRRS,        190        200        210        220        230        240 RVRRSS, YSTDNKYDAA GYSVDNENPL SGKAGGVVKV TYPGLTKVLA LKVDNAETIK KELGLSLTEP RVRRAT        250        260        270        280        290        300 were also LMEQVGTEEF IKRFGDGASR VVLSLPFAEG SSSVEYINNW EQAKALSVEL EINFETRGKR made.        310        320        330        340        350        360 GQDAMYEYMA QACAGN RVRR   AS VGSSLSCI NLDWDVIRDK TKTKIESLKE HGPIKNKMSE        370        380        390        400        410        420 SPNKTVSEEK AKQYLEEFHQ TALEHPELSE LKTVTGTNPV FAGANYAAWA VNVAQVIDSE        430        440        450        460        470        480 TADNLEKTTA ALSILPGIGS VMGIADGAVH HNTEEIVAQS IALSSLMVAQ AIPLVGELVD        490        500        510        520        530        540 IGFAAYNFVE SIINLFQVVH NSYNRPAYSP GHKTQPKGEL KLANIQLVQS GPELKKPGET        550        560        570       580         590        600 VKISCKASGY TFTNYGMNWV KQAPGKGLRW MGWINTHTGE PTYADDFKGR FAFSLETSAS        610        620        630        640        650        660 TAYLQINNLK NEDTATYFCT RRGYDWYFDV WGAGTTVTVF SGGGGSGGGG SGGGSSDIKM        670        680        690        700        710        720 TQSPSSMYAS LGERVTITCK ASQDINSYLS WFHHKPGKSP KTLIYRANRL VDGVPSRFSG        730        740        750        760        770        780 SGSGQDYSLT ISSLDYEDMG IYYCQQYDES PWTFGGGTKL ENKEQLLISE EDLGHHHHHH 115 DNA sequence for atgggatctgataaaattattcatctgactgatgattcttttgatactgatgtacttaaggcagatg Trx-DT^(A)-anti- gtgcaatcctggttgatttctgggcacactggtgcggtccgtgcaaaatgatcgctccgattctgga CD19 tgaaatcgctgacgaatatcagggcaaactgaccgttgcaaaactgaacatcgatcacaacccgggc actgcgccgaaatatggcatccgtggtatcccgactctctgctgttcaaaaacggtgaagtggcggc aaccaaagtgggtgcactgtctaaaggtcagttgaaagagttcctcgacgctaacctggccggctct ggatccggtgatgacgatgacaagctgggaattgatcccttcaccggcgccgacgacgtggtggact cctccaagtccttcgtcatggaaaacttcgcttcctaccacgggactaaacctggttatgtagattc cattcaaaaaggtatacaaaagccaaaatctggtacacaaggaaattatgacgatgattggaaaggg ttttatagtaccgacaataaatacgacgctgcgggatactctgtagataatgaaaacccgctctctg gaaaagctggaggcgtggtcaaagtgacgtatccaggactgacgaaggttctcgcactaaaagtgga taatgccgaaactattaagaaagagttaggtttaagtctcactgaaccgttgatggagcaagtcgga acggaagagtttatcaaaaggttcggtgatggtgcttcgcgtgtagtgctcagccttcccttcgctg aggggagttctagcgttgaatatattaataactgggaacaggcgaaagcgttaagcgtagaacttga gattaattttgaaacccgtggaaaacgtggccaagatgcgatgtatgagtatatggctcaagcctgt gccggcaatcgcgtgcgccgcgctagcgtggggagctcattgtcatgcatcaacctggactgggacg tgatccgcgacaagaccaagaccaagatcgagtccctgaaggagcacggcccgatcaagaacaagat gtccgagtccccgaacaagaccgtgtccgaggagaaggctaagcagtacctggaggagttccaccag accgctctggagcacccggagctgtccgagctgaaaaccgtgaccggcaccaacccggtgttcgctg gcgctaactacgctgcttgggctgtgaacgtggctcaggtgatcgactccgagactgctgacaacct ggagacaaccaccgctgctctgtccatcctgccgggcatcggctccgtgatgggcatcgctgacggc gctgtgcaccacaacaccgaggagatcgtggctcagtccatcgctctgtcctccctgatggtggctc aggctatcccgctggtgggcgagctggtggacatcggcttcgctgcttacaacttcgtggagtccat catcaacctgttccaggtggtgcacaactcctacaaccgcccggcttactccccgggccacaagacc cagcccaagggcgagctcaagcttgcccaggtgcagctgcagcagtccggcgctgagctggtgcgcc ctggctcctecgtgaaaatctcctgcaaggcttccggctacgctttctcctcctactggatgaactg ggtgaagcagcgccctggccagggcctggagtggatcggcccaatctggccgggcgacggcgccacc aactacaacggcaagttcaagggcaaggctaccctgaccgctgacgagtcctcctccaccgcttaca tgcagctgtcctccctggcttccgaggactccgctgtgtacttctgcgctcgccgcgagaccaccac cgtgggccgctactactacgctatggactactggggccagggcacctcggtgaccgtgtcctccggc ggcggcggctccggcggcggcggctccggcggcgggagctccgacatcctgctgacccagaccccgg cttccctggctgtgtccctgggccagcgcgctaccatctcctgcaaggcttcccagtccgtggacta cgacggcgactcctacctgaactggtaccagcagatcccgggccagccgccgaagctgctgatctac gacgcttccaacctggtgtccggcatcccgccgcgcttctccggctccggctccggcaccgacttca ccctgaacatccacccggtggagaaggtggacgctgctacctaccactgccagcagtccaccgagga cccgtggaccttcggcggcggcaccaagctggagatcaagcgcggtggtgacatgcatcaccatcac catcactgaagctt 116 Protein Sequence MGSDKIIHLTDDSFDTDVLKADGAILVDFWAHWCGPCKMIAPILDEIADEYQGKLTVAKLNIDHNPG for TrK-DT TAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFLDANLAGSGSGENLYFQLGIDPFTGADDVV (containing  DSSKSFVMENFASYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKGFYSTDNKYDAAGYSVDNENPL native SGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPF cell binding AEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRASVGSSLSCINLDW domain) DVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSEEKAKQYLEEFHQTALEHPELSELKTVTGTNPVF AGANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMV AQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKTQPKGELKLFLHDGYAVSWNTV EDSIIRTGFQGESGHSIKITAENTPLPIAGVLLPTIPGKLDVNKSKTHISVNGRKIRMRCRAIDGDV TFCRPKSPVYVGNGVHANLHVAFNRSSSEKIHSNEISSDSIGVLGYQKTVDHTKVNSKLSLFFEIKS KLEGKPIPNPLLGLDSTRTGHHHHHH 117 DNA Sequence ATGGCATCTGATAAAATTATTCATCTGACTGATGATTCTTTTGATACTGATGTACTTAAGGCAGATG for Trx-DT GTGCAATCCTGGTTGATTTCTGGGCACACTGGTGCGGTCCGTGCAAAATGATCGCTCCGATTCTGGA (containing TGAAATCGCTGACGAATATCAGGGCAAACTGACCGTTGCAAAACTGAACATCGATCACAACCCGGGC native ACTGCGCCGAAATATGGCATCCGTGGTATCCCGACTCTGCTGCTGTTCAAAAACGGTGAAGTGGCGG cell binding CAACCAAAGTGGGTGCACTGTCTAAAGGTCAGTTGAAAGAGTTCCTCGACGCTAACCTGGCCGGCTC domain) TGGATCCGGT GAA AAC CTG TAT TTT CAG GGC CTGGGAATTGATCCCTTCACC GGCGCCGACGACGTGGTGGACTCCTCCAAGTCCTTCGTCATGGAAAACTTCGCTTCCTACCACGGGA CTAAACCTGGTTATGTAGATTCCATTCAAAAAGGTATACAAAAGCCAAAATCTGGTACACAAGGAAA TTATGACGATGATTGGAAAGGGTTTTATAGTACCGACAATAAATACGACGCTGCGGGATACTCTGTA GATAATGAAAACCCGCTCTCTGGAAAAGCTGGAGGCGTGGTCAAAGTGACGTATCCAGGACTGACGA AGGTTCTCGCACTAAAAGTGGATAATGCCGAAACTATTAAGAAAGAGTTAGGTTTAAGTCTCACTGA ACCGTTGATGGAGCAAGTCGGAACGGAAGAGTTTATCAAAAGGTTCGGTGATGGTGCTTCGCGTGTA GTGCTCAGCCTTCCCTTCGCTGAGGGGAGTTCTAGCGTTGAATATATTAATAACTGGGAACAGGCGA AAGCGTTAAGCGTAGAACTTGAGATTAATTTTGAAACCGGTGGAAAACGTGGCCAAGATGCGATGTA TGAGTATatggctcaagcctgtgccggcAATcgcgtgcgccgcGCTagcgtggggagctcattgtca TGCATCAACCTGGACTGGGACGTGATCCGCGACAAGACCAAGACCAAGATCGAGTCCCTGAAGGAGC ACGGCCCGATCAAGAACAAGATGTCCGAGTCCCCGAACAAGACCGTGTCCGAGGAGAAGGCTAAGCA GTACCTGGAGGAGTTCCACCAGACCGCTCTGGAGCACCCGGAGCTGTCCGAGCTGAAAACCGTGACC GGCACCAACCCGGTGTTCGCTGGCGCTAACTACGCTGCTTGGGCTGTGAACGTGGCTCAGGTGATCG ACTCCGAGACTGCTGACAACCTGGAGAAAACCACCGCTGCTCTGTCCATCCTGCCGGGGATCGGCTC CGTGATGGGCATCGCTGACGGCGCTGTGCACCACAACACCGAGGAGATCGTGCCTCAGTCCATCGCT CTGTCCTCCCTGATGGTGGCTCAGGCTATCCCGCTGGTGGGCGAGCTGGTGGACATCGGCTTCGCTG CTTACAACTTCGTGGAGTCCATCATCAACCTGTTCCAGGTGGTGCACAACTCCTACAACCGCCCGGC TTACTCCCCGGGCCACAAGACCCAGCCC AAGGGCGAGCTCAAGCTTTTTCTTCATGACGGGTATGCTGTCAGTTGGAACACTGTTGAAGATTCGA TAATCCGAACTGGTTTTCAAGGGGAGAGTGGGCACGACATAAAAATTACTGCTGAAAATACCCCGCT TCCAATCGCGGGTGTCCTACTACCGACTATTCCTGGAAAGCTGGACGTTAATAAGTCCAAGACTCAT ATTTCCGTAAATGGTCGGAAAATAAGGATGCGTTGCAGAGCTATAGACGGTGATGTAACTTTTTGTC GCCCTAAATCTCCTGTTTATGTTCGTAATGGTGTGCATGCGAATCTTCACGTGGCATTTCACAGAAG CAGCTCGGAGAAAATTCATTCTAATGAAATTTCGTCGGATTCCATAGGCGTTCTTGGGTACCAGAAA ACAGTAGATCACACCAAGGTTAATTCTAAGCTATCGCTATTTTTTGAAATCAAAAGCAAGCTT 118 DT-anti-CD2219 MGADDVVDSS KSFVMENFAS YHGTKPGYVD SIQKGIQKPK SGTQGNYDDD WKGFYSTDNK protein sequence YDAAGYSVDN ENPLSGKAGG VVKVTYPGLT KVLALKVDNA ETIKKELGLS LTEPLNEQVG TEEFIKRFGD GASRVVLSLP FAEGSSSVEY INNWEQAKAL SVELEINFET RGKRGQDAMY EYNAQACAGN IEPDTGSSLS CINLDWDVIR DKTKTKIESL KEHGPIKNKM SESPAKTVSE EKAKQYLEEF HQTALEHPEL SELKTVTGTN PVFAGANYAA WAVNVAQVID SETADNLEKT TAALSILPGI GSVMGIADGA VHHNTEEIVA QSIALSSLMV AQAIPLVGEL VDIGFAAYNF VESIINLFQV VHNSYNRPAY SPGHKTQPAM EVQLVESGGG LVKPGGSLKL SCAASGFAFS IYDMSWVRQT PEKRLEWVAY ISSGGGTTYY PDTVKGRFTI SRDNAKNTLY LQMSSLKSED TAMYYCARHS GYGTHWGVLF AYWGQGTLVT VSAGGGGSGG GGSGGGSSDI QMTQTTSSLS ASLGDRVTIS CRASQDIARY LNWYQQKPDG TVKLLIYYTS ILHSGVFSRF SGSGSGTDYS LTISNLEQED FATYFCQQGN TLPWTFGGGT KLEIKTGPSG QAGAAASESL FVSNHAYTMA QVQLQQSGAE LVRPGSSVKI SCKASGYAFS SYWMNWVKQR PGQGLEWIGQ IWPGOGDTNY NGKFKGKATL TADESSSTAY MQLSSLASED SAVYFCARRE TTTVGRYYYA MDYWGQGTSV TVSSGGGGSG GGGSGGGSSD ILLTQTPASL AVSLGQRATI SCKASQSVDY DGDSYLNWYQ QIPGQPPKLL IYDASNLVSG IPPRFSGSGS GTDFTLNIHP VEKVDAATYH CQQSTEDPWT FGGGTKLEIK RGGDMHHHHH H 119 DT-anti-CD2219 ATGGGTGCCGACGACGTGGTGGACTCCTCCAAGTCCTTCGTGATGGAAAACTTCGCTTCCTACCACG DNA sequence GTACCAAGCCTGGTTACGTGGATTCCATCCAGAAGGGTATCCAGAAGCCTAAGTCCGGTACCCAGGG TAACTACGACGATGATTGGAAGGGTTTTTACTCCACCGACAACAAGTACGACGCCGCCGGTTACTCC GTGGATAACGAAAACCCTCTGTCCGGTAAGGCCGGTGGTGTGGTGAAAGTGACCTACCCTGGTCTGA CCAAGGTGCTGGCCCTGAAGGTGGATAACGCCGAAACCATCAAGAAGGAGCTGGGTCTGTCCCTGAC CGAACCTCTGATGGAGCAGGTGGGTACCGAAGAGTTTATCAAGAGATTCGGTGATGGTGCCTCCAGA GTGGTGCTGTCCCTGCCTTTCGCCGAGGGTTCCTCCTCCGTGGAATACATCAACAACTGGGAACAGG CCAAGGCCCTGTCCGTGGAACTGGAGATCAACTTTGAAACCAGAGGTAAGAGAGGTCAGGATGCCAT GTACGAGTACatggcccaggcctgtgccggCAACATCGAGCCTGACACCGgttcctccctgtccTGC ATCAACCTGGACTGGGACGTGATCAGAGACAAGACCAAGACCAAGATCGAGTCCCTGAAGGAGCACG GTCCTATCAAGAACAAGATGTCCGAGTCCCCTGCCAAGACCGTGTCCGAGGAGAAGGCCAAGCAGTA CCTGGAGGAGTTCCACCAGACCGCCCTGGAGCACCCTGAGCTGTCCGAGCTGAAGACCGTGACTGGT ACCAACCCTGTGTTCGCCGGTGCCAACTACGCCGCCTGGGCCGTGAACGTGGCCCAGGTGATCGACT CCGAGACCGCCGACAACCTGGAGAAGACCACCGCCGCCCTGTCCATCCTGCCTGGTATCGGTTCCGT GATGGGTATCGCCGACGGTGCCGTCCACCACAACACCGAGGAGATCGTGGCCCAGTCCATCGCCCTG TCCTCCCTGATGGTGGCCCAGGCCATCCCTCTGGTGGGTGAGCTGGTGGACATCGGTTTCGCCGCCT ACAACTTCGTGGAGTCCATCATCAACCTGTTCCAGGTGGTGCACAACTCCTACAACAGACCTGCCTA CTCCCCTGGTCACAAGACCCAGCCTGccATGGAGGTTCAGCTGGTTGAGTCCGGTGGTGGTCTGGTT AAGCCAGGTGGTTCCCTGAAGCTGTCCTGTGCTGCTTCCGGTTTCGCTTTCTCCATCTACGATATGT CCTGGGTTAGACAGACCCCAGAGAAGAGACTGGAGTGGGTTGCTTACATCTCCTCCGGTGGTGGTAC CACCTACTACCCAGACACCGTTAAGGGTAGATTCACCATCTCCAGAGATAACGCTAAGAACACCCTG TACCTGCAGATGTCCTCCCTGAAGTCCGAGGACACCGCTATGTACTACTGTGCTAGACATTCCGGTT ACGGTACCCATTGGGGTGTTCTGTTCGCTTACTGGGGTCAGGGTACCCTGGTTACCGTTTCCGCTGG TGGTGGTGGTTCCGGTGGTGGTGGTTCCGGTGGTGGGAGCTCCGATATCCAGATGACCCAGACCACC TCCTCCCTGTCCGCTTCCCTGGGTGACAGAGTTACCATCTCCTGTAGAGCTTCCCAGGATATCGCTA GATACCTGAACTGGTACCAGCAGAACCCAGACGGTACCGTTAAGCTGCTGATCTACTACACCTCCAT CCTGCATTCCGGTGTTCCATCCAGATTCTCCGGTTCCGGTTCCGGTACCGATTACTCCCTGACCATC TCCAACCTGGAGCAGGAGGACTTCGCTACCTACTTCTGTCAGCAGGGTAACACCCTGCCTTGGACCT TCGGTGGTGGTACCAAGCTGGAGATCAAGACTGGTCCATCCGGTCAGGCTGGTGCTGCTgctTCCGA GTCCTTGTTCGTTTCCAACCACGCTTACACCATGGCCCAGGTTCAGTTGCAGCAGTCCGGTGCTGAG TTGGTTAGACCAGGTTCCTCTGTTAAGATCTCTTGTAAGGCCTCTGGCTATGCTTTTTCCTCTTACT GGATGAACTGGGTTAAGCAGAGACCAGGTCAGGGCTTGGAATGGATCGGTCAAATTTGGCCAGGTGA TGGTGATACTAACTACAACGGTAAGTTCAAGGGTAAGGCTACTTTGACTGCTGACGAATCCTCCTCT ACTGCCTATATGCAACTGTCCTCTCTGGCTTCTGAAGATTCTGCTGTTTACTTCTGCGCTAGAAGAG AAACCACTACCGTTGGTAGATACTACTATGCTATGGATTACTGGGGTCAAGGTACCTCGGTGACCGT TTCTTCCGGTGGCGGTGGTTCTGGTGGTGGTGGCTCTGGTGGTGGGAGCTCCGATATCTTGTTGACT CAAACCCCAGCTTCTTTGGCTGTGTCTCTGGGTCAAAGAGCTACTATTTCCTGCAAGGCTTCTCAAT CTGTGGATTACGATGGTGACTCCTACTTGAATTGGTATCAGCAGATTCCAGGTCAGCCTCCTAAGCT GTTGATCTACGATGCTTCCAACTTGGTCTCCGGTATCCCACCAAGATTCTCCGGTTCTGGTTCCGGT ACTGACTTCACTTTGAACATCCACCCAGTTGAGAAAGTGGATGCTGCCACTTACCACTGCCAACAAT CTACCGAGGATCCTTGGACTTTCGGTGGTGGTACCAAGTTGGAGATCAAAAGAGGTGGTGACATGCA CCATCACCACCACCATTAA 120 GrB-anti-CD1919 IIGGHEAKPR SRPYMAYLMI WDQKSLKRCG GFLIQDDFVL TAAHCWGSSI NVTLGAHNIK protein sequence EQEETQQFIP VKRPIPHPAY NPKNFSNDIM LLQLERKAKR TRAVQPLRLP SNKAQVKPGQ TCSVAGWGQT APLGKHSHTL QEVKMTVQED RKCESDLRHY YDSTIELCVG DPEIKKTSFK GDSGGPLVCN KVAQGIVSYG RNNGMPPRAC TKVSSFVHWI KKTMKRYPNG GGGSGGGGSG GGGSAQVQLQ QSGAELVRFG SSVKISCKPS GYAFSSYWMN WVKQRPGQGL EWIGQIWPGD GDTNYNGKFK GKATLTADES SSTAYMQLSS LASEOSAVYF CARRETTTVG RYYYAMDYWG QGTSVTVSSG GGGSGGGGSG GGSSDILLTQ TPASLAVSLG QRATISCKAS QSVDYDGDSY LNWYQQIPGQ PPKLLIYDAS NLVSGIPPRF SGSGSGTDFT LNIHPVEKVD AATYHCQQST EDPWTEGGGT KLEIKRGGDM GNSGGGGAQV QLQQSGAELV RPGSSVKISC KASGYAFSSY WMNWVKQRPG QGLEWIGQIW PGDGDTNYNG KFKGKATLTA DESSSTAYMQ LSSLASEDSA VYFCARRETT TVGRYYYAND YWGQGTSVTV SSGGGGSGGG GSGGGSSDIL LTQTPASLAV SLGQRATISC KASQSVDYDG DSYLNWYQQI PGQPPKLLIY DASNLVSGIP PRFSGSGSGT DFTLNIHPVE KVOAATYHCQ QSTEDPWTFG GGTKLEIKRG GDMHHHHHH 121 GrB-anti-CD1919 atcatcgggggacatgaggccaagccccactcccgcccctacatggcttatettatgatctgggatc DNA sequence agaagtctctgaagaggtgcggtggcttcctgatacaagacgacttcgtgctgacagctgctcactg ttggggaagctccataaatgtcaccttgggggcccacaatatcaaagaacaggagccgacccagcag tttatccctgtgaaaagacccatcccccatccagcctataatcctaaggacttctccaacgacatca tgctactgcagctggagagaaaggccaagcggaccagagctgtgcagcccctcaggctacctagcaa caaggcccaggtgaagccagggcagacatgcagtgtggccggctgggggcagacggcccccctggga aaacactcacaCacactacaagaggtgaggattacagtgcaggaagatcgaaagtgcgaatctgact tacgccattattacgacagtaccattgagttgtgcgtgggggacccagagattaaaaagacttcctt taagggggactctggaggccctcttgtgtgtaacaaggtggcccagggcattgtctcctatggacga aacaatggcatgcctccacgagcctgcaccaaagtctcaagctttgtacactggataaagaaaacca tgaaacgctacgccATGGGAGGCGGAGGCTCCGGAGGAGGAGGGTCCGGGGGCGGCGGAAGCGCCCA GGTTCAGTTGCAGCAGTCCGGTGCTGAGTTGGTTAGACCAGGTTCCTCTGTTAAGATCTCTTGTAAG GCCTCTGGCTATGCTTTTTCCTCTTACTGGATGAACTGGGTTAAGCAGAGACCAGGTCAGGGCTTGG AATGGATCGGTCAAATTTGGCCAGGTGATGCTGATACTAACTACAACGGTAAGTTCAAGGGTAAGGC TACTTTGACTGCTGACGAATCCTCCTCTACTGCCTATATGCAACTGTCCTCTCTGGCTTCTGAAGAT TCTGCTGTTTACTTCTGCGCTAGAAGAGAAACCACTACCGTTGGTAGATACTACTATGCTATGGATT ACTGGGGTCAAGGTACCTCGGTGACCGTTTCTTCCGGTGGCGGTGGTTCTGGTGGTGGTGGCTCTGG TGGTGGGAGCTCCGATATCTTGTTGACTCAAACCCCAGCTTCTTTGGCTGTGTCTCTGGGTCAAAGA GCTACTATTTCCTGCAAGGCTTCTCAATCTGTGGATTACGATGGTGACTCCTACTTGAATTGGTATC AGCAGATTCCAGGTCAGCCTCCTAAGCTGTTGATCTACGATGCTTCCAACTTGGTCTCCGGTATCCC ACCAAGATTCTCCGGTTCTGGTTCCGGTACTGACTTCACTTTGAACATCCACCCAGTTGAGAAAGTG GATGCTGCCACTTACCACTGCCAACAATCTACCGAGGATCCTTGGACTTTCGGTGGTGGTACCAAGT TGGAGATCAAAAGAGGTGGTGACATGGggaattctGGAGGCGGAGGCGCCCAGGTTCAGTTGCAGCA GTCCGGTGCTGAGTTGGTTAGACCACGTTCCTCTGTTAAGATCTCTTGTAAGGCCTCTGGCTATGCT TTTTCCTCTTACTGGATGAACTGGGTTAAGCAGAGACCAGGTCAGGGCTTGGAATGGATCGGTCAAA TTTGGCCAGGTGATGGTGATACTAACTACAACGGTAAGTTCAAGGGTAAGGCTACTTTGACTGCTGA CGAATCCTCCTCTACTGCCTATATGCAACTGTCCTCTCTGGCTTCTGAAGATTCTGCTGTTTACTTC TGCGCTAGAAGAGAAACCACTACCGTTGGTAGATACTACTATGCTATGGATTACTGGGGTCAAGGTA CCTCGGTGACCGTTTCTTCCGGTGTCGGTGGTTCTGGTGGTGGTGGCTCTGGTGGTGGGAGCTCCGA TATCTTGTTGACTCAAACCCCAGCTTCTTTGGCTGTGTCTCTGGGTCAAAGAGCTACTATTTCCTGC AAGGCTTCTCAATCTGTGGATTACGATGGTGACTCCTACTTGAATTGGTATCAGCAGATTCCAGGTC AGCCTCCTAAGCTGTTGATCTACGATGCTTCCAACTTGGTCTCCGGTATCCCACCAAGATTCTCCGG TTCTGGTTCCGGTACTGACTTCACTTTGAACATCCACCCAGTTGAGAAAGTGGATGCTGCCACTTAC CACTGCCAACAATCTACCGAGGATCCTTGGACTTTCGGTGGTGGTACCAAGTTGGAGATCAAAAGAG GTGGTGACATGCACCATCACCACCACCATTAAGC 122 MBP-GKGgGS- MFPSHMKTEE GKLVIWINGD KGYNGLAEVG KKFEKDTGIK VTVEHPDKLE EKFPQVAATG TEV protein DGPDIIFWAH DRFGGYAQSG LLAEITPDKA FQDKLYPFTW DAVRYNGKLI AYPIAVEALS sequence LIYNKDLLPN PPKTWEEIPA LDKELKAKGK SALMFNLQEP YFTWPLIAAD GGYAFKYENG KYDIKDVGVD NAGAKAGLTF IVDLIKNKHM NADTDYSIAE AAFNKGETAM TINGPWAWSN IDTSKVNYGV TVLPTFKGQP SKPFVGVLSA GINAASPNKE LAKEFLENYL LTDEGLEAVN KDKPLGAVAL KSYEEELAKD PRIAATMENA QKGEIMPNIP QMSAFWYAVR TAVINAASGR QTVDEALKDA QTNSSNNSRR ASVAMLRQIL DSQKMEWRSN AMTGGGSKLG DDDDKGKGGG SKGPRDYNPI SSAICHLTNE SDGHTTSLYG IGFGPFIITN KHLFRRNNGT LLVQSLHGVF KVKNTTTLQQ HLIDGRDMML IRMPKDFPPF PQKLKFREPQ REERICLVTT NFQTKSMSSM VSDTSCTFPS SDGIFWKHWI QTKDGHCGSP LVSTRDGFIV GIHSASNFTN TNNYFTSVPK DFMDLLTNQE AQQWVSGWRL NADSVLWGGH KVFMNKPEEP FQPVKEATQL MSHHHHHH 123 MBP-GKGGGS- atgccaccctcccatATGAAAACTGAAGAAGGTAAACTGGTAATCTGGATTAACGGCGATAAAGGCT TEV DNA ATAACGGTCTCGCTGAAGTCGGTAAGAAATTCGAGAAAGATACCGGAATTAAAGTCACCGTTGAGCA sequence TCCGGATAAACTGGAAGAGAAATTCCCACAGGTTGCGGCAACTGGCGATGGCCCTGACATTATCTTC TGGGCACACGACCGCTTTGGTGGCTACGCTCAATCTGGCCTGTTGGCTGAAATCACCCCGGACAAAG CGTTCCAGGACAAGCTGTATCCGTTTACCTGGGATGCCGTACGTTACAACGGCAAGCTGATTGCTTA CCCGATCGCTGTTGAAGCGTTATCGCTGATTTATAACAAAGATCTGCTGCCGAACCCGCCAAAAACC TGGGAAGAGATCCCGGCGCTGGATAAAGAACTGAAAGCGAAAGGTAAGAGCGCGCTGATGTTCAACC TGCAAGAACCGTACTTCACCTGGCCGCTGATTGCTGCTGACGGGGGTTATGCGTTCAAGTATGAAAA CGGCAAGTACGACATTAAAGACGTGGGCGTGGATAACGCTGGCGCGAAAGCGGGTCTGACCTTCCTG GTTGACCTGATTAAAAACAAACACATGAATGCAGACACCGATTACTCCATCGCAGAAGCTGCCTTTA ATAAAGGCGAAACAGCGATGACCATCAACGGCCCGTGGGCATGGTCCAACATCGACACCAGCAAAGT GAATTATGGTGTAACGGTACTGCCGACCTTCAAGGGTCAACCATCCAAACCGTTCGTTGGCGTGCTG AGCGCAGGTATTAACGCCGCCAGTCCGAACAAAGAGCTGGCAAAAGAGTTCCTCGAAAACTATCTGC TGACTGATGAAGGTCTGGAAGCGGTTAATAAAGACAAACCGCTGGGTGCCGTAGCGCTGAAGTCTTA CGAGGAAGAGTTGGCGAAAGATCCACGTATTGCCGCCACTATGGAAAACGCCCAGAAAGGTGAAATC ATGCCGAACATCCCGCAGATGTCCGCTTTCTGGTATGCCGTCCGTACTGCGGTGATCAACGCCGCCA GCGGTCGTCAGACTGTCGATGAAGCCCTGAAAgacgcgcagactaattcgagcaacaactcacggcg ggctagtgtcgccatgctgcgtcaaattctggattctcaaaaaatggaatggcgctctaacgccatg accggtGGCGGGAGCaagcttggggatgacgatgacaagggcaaaGGCGGCGGGAGCAAAGGTCCGC GTGACTACAACCCGATCTCCTCCGCTATCTGCCACCTGACCAACGAATCCGACGGTCACACCACCTC CCTGTACGGTATCGGTTTCGGTCCGTTCATCATCACCAACAAACACCTGTTCCGTCGTAACAACGGG ACCCTGCTGGTTCAGTCCCTGCACGGTGTTTTCAAAGTTAAAAACACCACCACCCTCCAGCAGCACC TGATCGACGGTCGTGACATGATGCTGATCCGTATGCCGAAAGACTTCCCGCCGTTCCCGCAGAAACT GAAATTCCGTGAACCGCAGCGTGAAGAACGTATCTGCCTCGTTACCACCAACTTCCAGACCAAATCC ATGTCCTCTATGGTTTCCGACACCTCCTGCACCTTCCCGTCCTCCGACGGTATCTTCTGGAAACACT GGATTCAGACCAAAGACGGTCACTGCGGTTCCCCGCTGGTTTCCACCCGTGACGGTTTCATCGTTGG TATCCACTCCGCTTCCAACTTCACCAACACCAACAACTACTTCACCTCCGTTCCGAAAGACTTCATG GACCTCCTGACCAACCAGGAAGCTCAGCAGTGGGTTTCCGGTTGGCGTCTGAACGCTGACTCCGTTC TGTGGGGTGGTCACAAAGTTTTTATGAACAAACCGGAAGAACCGTTCCAGCCGGTTAAAGAAGCTAC CCAGCTCATGTCCCACCATCACCACCACCATtaagcggccgcgaattc 124 GrM-anti-CD19 IIGGREVIP HSRPYMASLQ RNGSHLCGGV LVHPKWVLTA AHCLAQRMAQ LRLVLGLHTL protein sequence DSPGLTFHIK AAIQHPRYKP VPALEWDLAL LQLDGKVKPS RTIRPLALPS KRQVVAAGTR CSMAGWGLTH QGGRLSRVLR ELDLQVLDTR MCNNSRFWNG SLSPSMVCLA ADSKDQAPCK GDSGG2LVCG KGRVLAGVLS FSSRVCTDIF KPPVATAVAP YVSWIRKVTG RSAAMAQVQL QQSGAELVRP GSSVKISCKA SGYAFSSYWM NWVKQRPGQG LEWIGQIWPG DGDTNYNGKF KGKATLTADE SSSTAYMQLS SLASEDSAVY FCARRETTTV GRYYYAMDYW GQGTSVTVSS GGGGSGGGGS GGGSSDILLT QTPASLAVSL GQRATISCKA SQSVDYDGDS YLNWYQQIPG QPPKLLIYDA SNLVSGIPPR FSGSGSGTDF TLNIHPVEKV DAATYHCQQS TEDPWTFGGG TKLEIKRGGD MHHHHHH 125 GrM-anti-CD19 ctcgagctacaaggacgacgacgacaagatcatcgggggccgggaggtgatcccccactcgcgcccg DNA sequence tacatggcctcactgcagagaaatggctcccacctgtgcgggggtgtcctggtgcacccaaagtggg tgctgacggctgcccactgcctggcccagcggatggcccagctgaggctggtgctggggctccacac cctggacagccccggtctcaccttccacatcaaggcagccatccagcaccctcgctacaagcccgtc cctgccctggagaacgacctcgcgctgcttcagctggacgggaaagtgaagcccagccggaccatcc ggccgttggccctgcccagtaagcgccaggtggtggcagcagggactcggtgcagcatggccggctg ggggctgacccaccagggcgggcgcctgtcccgggtgctgcgggagctggacctccaagtgctggac acccgcatgtgtaacaacagccgcttctggaacggcagcctctcccccagcatggtctgcctggcgg ccgactccaaggaccaggctccctgcaagggtgactcgggcgggcccctggtgtgtggcaaaggccg ggtgttggccggagtcctgtccttcagctccagggtctgcactgacatcttcaagcctcccgtggcc accgctgtggcgccttacgtgtcctggatcaggaaggtcaccggccgatcggccgccatggccCAGG TGCAGCTGCAGCAGTCCGGCGCTGAGCTGGTGCGCCCTGGCTCCTCCGTGAAAATCTCCTGCAAGGC TTCCGGCTACGCTTTCTCCTCCTACTGGATGAACTGGGTGAAGCAGCGCCCTGGCCAGGGCCTGGAG TGGATCGGCCAAATCTGGCCGGGCGACGGCGACACCAACTACAACGGCAAGTTCAAGGGCTAGGCTA CCCTGACCGCTGACGAGTCCTCCTCCACCGCTTACATGCAGCTGTCCTCCCTGGCTTCCGAGGACTC CGCTGTGTACTTCTGCGCTCGCCGCGAGACCACCACCGTGGGCCGCTACTACTACGCTATGGACTAC TGGGGCCAGGGCACCTCGGTGACCGTGTCCTCCGGCGGCGGCGGCTCCGGCGGCGGCGGCTCCGGCG GCGGGAGCTCCGACATCCTGCTGACCCAGACCCCGGCTTCCCTGGCTGTGTCCCTGGGCCAGCGCGC TACCATCTCCTGCAAGGCTTCCCAGTCCGTGGACTACGACGGCGACTCCTACCTGAACTGGTACCAG CAGATCCCGGGCCAGCCGCCGAAGCTGCTGATCTACGACGCTTCCAACCTGGTGTCCGGCATCCCGC CGCGCTTCTCCGGCTCCGGCTCCGGCACCGACTTCACCCTGAACATCCACCCGGTGGAGAAGGTGGA CGCTGCTACCTACCACTGCCAGCAGTCCACCGAGGACCCGTGGACCTTCGGCGGCGGCACCAAGCTG GAGATCAAGCGCggtggtgacatgCACCATCACCACCACCATTAAGC 126 PP2C-anti-CD5 ATGGGATCTGATAAAATTATTCATCTGACTGATGATTCTTTTGATACTGATGTACTTAAGGCAGATG scFv DNA GTGCAATCCTGGTTGATTTCTGGGCACACTGGTGCGGTCCGTGCAAAATGATCGCTCCGATTCTGGA sequence TGAAATCGCTGACGAATATCAGGGCAAACTGACCGTTGCAAAACTGAACATCGATCACAACCCGGGC ACTGCGCCGAAATATGGCATCCGTGGTATCCCGACTCTGCTGCTGTTCAAAAACGGTGAAGTGGCGG CAACCAAAGTGGGTGCACTGTCTAAAGGTCAGTTGAAAGAGTTCCTCGACGCTAACCTGGCCGGCTC TGGATCCGGTGATGACGATGACAAGCTGGGAATTGATCCCTTCACCATGGGAGCATTTTTAGACAAG CCAAAGATGGAAAAGCATAATGCCCAGGGGCAGGGTAATGGGTTGCGATATGGGCTAAGCAGCATGC AAGGCTGGCGTGTTGAAATGGAGGATGCACATACGGCTGTGATCGGTTTGCCAAGTGGACTTGAATC GTGGTCATTCTTTGCTGTGTATGATGGGCATGCTGGTTCTCAGGTTGCCAAATACTGCTGTGAGCAT TTGTTAGATCACATCACCAATAACCAGGATTTTAAAGGGTCTGCAGGAGCACCTTCTGTGGAAAATG TAAAGAATGGAATCAGAACAGGTTTTCTGGAGATTGATGAACACATGAGAGTTATGTCAGAGAAGAA ACATGGTGCAGATAGAAGTGGGTCAACAGCTGTAGGTGTCTTAATTTCTCCCCAACATATACTTATT TCATTAACTGTGGAGACTCAAGAGGTTACTTTGTAGGAACAGGAAAGTTCATTTCTTCACACAAGAT CACAACCAAGTAATCCGCTGGAGAAAGAACGAATTCAGAATGCAGGTGGCTCTGTAATGATTCAGCG TGTGAATGGCTCTCTGGCTGTATCGAGGGCCCTTGGGGATTTTGATTACAAATGTGTCCATGGAAAA GGTCCTACTGAGCAGCTTGTCTCACCAGAGCCTGAAGTCCATGATATTGAAAGATCTGAAGAAGATG ATCAGTTCATTATCCTTGCATGTGATGGTATCTGGGATGTTATGGGAAATGAAGAGCTCTGTGATTT TGTAAGATCCAGACTTGAAGTCACTGATGACCTTGAGAAAGTTTGCAATGAAGTAGTCGACACCTGT TTGTATAAGGGAAGTCGAGACAACATGAGTGTGATTTTGATCTGTTTTCCAAATGCACCCAAAGTAT CGCCAGAAGCAGTGAAGAAGGAGGCAGAGTTGGACAAGTACCTGGAATGCAGAGTAGAAGAAATCAT AAAGAAGCAGGGGGAAGGCGTCCCCGACTTAGTCCATGTGATGCGCACATTAGCGAGTGAGAACATC CCCAGCCTCCCACCAGGGGGTGAATTGGCAAGCAAGAGGAATGTTATTGAAGCCGTTTACAATAGAC TGAATCCTTACAAAAATGACGACACTGACTCTACATCAACAGATGATATGTGGAAGGGCGAGCTCAA GCTTGCCAACATCCAGCTGGTGCAGTCTGGTCCTGAGCTGAAGAAGCCTGGTGAGACTGTCAAAATC TCCTGCAAGGCTTCTGGGTATACCTTCACTAACTATGGTATGAACTGGGTGAAGCAGGCTCCTGGTA AGGGTCTGCGTTGGATGGGCTGGATIAACACCCACACTGGTGAGCCTACTTATGCTGATGACTTCAA GGGACGTTTTGCCTTCTCTCTGGAAACTTCTGCCAGCACTGCCTATCTCCAGATCAACAACCTCAAA AATGAGGACACTGCTACTTACTTCTGTACACGTCGTGGTTACGACTGGTACTTCGATGTCTGGGGTG CTGGGACCACGGTGACCGTGTTCTCCGGGGGAGGTGGCAGCGGGGGAGGTGGCAGCGGCGGCGGGAG CTCCGACATCAAGATGACCCAGTCTCCTTCTTCCATGTATGCTTCTCTGGGTGAGCGTGTCACTATC ACTTGCAAGGCCAGCCAGGACATTAATAGCTATCTGAGCTGGTTCCATCATAAACCTGGGAAATCTC CTAAGACCCTGATCTATCGTGCTAACCGTCTGGTTGATGGGGTCCCTTCTCGTTTCAGCGGCTCTGG TTCTGGGCAAGATTATTCTCTCACCATCAGCAGCCTGGACTATGAAGATATGGGTATTTATTATTGT CAACAGTATGATGAGTCTCCTTGGACTTTCGGTGGTGGCACCAAGCTGGAGATGAAAGAACAAAAGT TGATCTCCGAAGAGGATTTGGGTCATCATCACCATCACCATTAAGCGGCCGCATAAGCTT 127 PP2C-anti-CD5         10         20         30         40         50         60 scFv protein MGSDKIIHLT DDSFDTDVLK ADGAILVDFW AHWCGPCKMI APILDEIADE YQGKLTVAKL sequence         70         80         90        100        110        120 NIDHNPGTAP KYGIRGIPTL LLFKNGEVAA TKVGALSKGQ LKEFLDANLA GSGSGDDDDK        130        140        150        160        170        180 LGIDPFTNDA FLDKPKMEKH NAQGQGNGLR YGLSSMQGWR VEMEDANTAV IGLPSGLESW        190        200        210        220        230        240 SFFAVYDGRA GSQVAKYCCE HLLDHITNNQ DFKGSAGAPS VENVKNGIRT GFLEIDEHMR        250        260        270        280        290        300 VMSEKKHGAD RSGSTAVGVL ISPQHTYFIN CGDSRGLLCR NRKVHFFTQD HKPSNPLEKE        310        320        330        340        350        360 RIQNAGGSVM IQRVNGSLAV SRALGDFDYK CVHGKGPTEQ LVSPEPEVHD IERSEEOOQF        370        380        390        400        410        420 IILACDGIWD VMGNEELCDF VRSRLEVTDD LEKVCNEVVD TCLYKGSRDN MSVILICFPN        430        440        450        460        470        480 APKVSPEAVK KEAELDKYLE CRVEEIIKKQ GEGVPDLVHV MRTLASENIP SLPPGGELAS        490        500        510        520        530        540 KRNVIEAVYN RLNPYKNDDT DSTSTDDMWK GELKLANIQL VQSGPELKKP GETVKISCKA        550        560        570        580        590        600 SGYTFTNYGM NWVKQAPGKG LRWMGWINTH TGEPTYADDF KGRFAFSLET SASTAYLQIN        610        620        630        640        650        660 NLKNEDTATY FCTRRGYDWY FDVWGAGTTV TVFSGGGGSG GGGSGGGSSD IKMTQSFSSM        670        680        690        700        710        720 YASLGERVTI TCKASQDINS YLSWFHHKPG KSPKTLIYRA NRLVDGVPSR FSGSGSGQDY        730        740        750        760        770 SLTISSLDYE DMGIYYCQQY DESPWTFGGG TKLEMKEQKL ISEEDLGHHN HHH

OTHER EMBODIMENTS

All publications, patent applications, and patents mentioned in this specification are herein incorporated by reference.

Various modifications and variations of the described method and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific desired embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the fields of medicine, pharmacology, or related fields are intended to be within the scope of the invention. 

What is claimed is:
 1. A composition comprising; (i) a protoxin fusion protein comprising a first non-native cell-targeting moiety, a selectively modifiable activation domain and a toxin domain; and a (ii) protoxin activator fusion protein comprising a second non-native cell-targeting moiety and a modification domain; wherein: said first cell-targeting moiety of said protoxin fusion protein and said second cell-targeting moiety of said protoxin activator fusion protein each recognize and bind a common target cell; said modification domain comprises protease or phosphatase enzymatic activity exogenous to said target cell; said selectively modifiable activation domain comprises a substrate for said modification domain; and modification of said selectively modifiable activation domain by said modification domain results in activation of said toxin domain.
 2. The composition of claim 1, wherein said enzymatic activity is protease activity.
 3. The composition of claim 1, wherein said modification domain is a phosphatase and said modifiable activation domain comprises phosphorylation of a protease cleavage site.
 4. The composition of claim 1, wherein at least one non-native cell-targeting moiety is an artificially diversified binding protein.
 5. The composition of claim 1, wherein said protoxin is an activatable toxin.
 6. The composition of claim 5, wherein said activatable toxin is selected from the group consisting of an activatable pore forming toxin or an activatable enzymatic toxin.
 7. The composition of claim 1, wherein said toxin domain is selected from a group consisting of an AB toxin, a cyotoxic necrotizing factor toxin, a dermonecrotic toxin, and an activatable ADP-ribosylating toxin.
 8. The composition of claim 1, wherein said toxin domain is selected from a group consisting of aerolysin, Vibrio cholerae exotoxin, Pseudomonas exotoxin and diphtheria toxin.
 9. The composition of claim 1, wherein said protoxin activator fusion protein further comprises a natively activatable domain wherein said modification domain is inactive prior to activation of said natively activatable domain and, when active, is non-toxic to a target cell.
 10. The composition of claim 1, wherein said modification domain is a protease domain.
 11. The composition of claim 10, wherein said protease domain is the catalytic domain of a non-human protease.
 12. The composition of claim 11, wherein said non-human protease is a viral protease.
 13. The composition of claim 1, wherein said non-native cell-targeting moiety recognizes a cancer cell.
 14. The composition of claim 1, wherein at least one non-native cell-targeting moiety is an antibody or antibody fragment.
 15. The composition of claim 1, wherein both of said cell-targeting moieties is an antibody or antibody fragment.
 16. The composition of claim 10, wherein said protease domain is the catalytic domain of an exogenous human protease.
 17. A composition comprising: (i) a protoxin fusion protein comprising a first non-native cell-targeting moiety, a selectively modifiable activation domain and a toxin domain; and a (ii) protoxin activator fusion protein comprising a second non-native cell-targeting moiety and a modification domain; wherein: said first cell-targeting moiety of said protoxin fusion protein and said second cell-targeting moiety of said protoxin activator fusion protein each recognize and bind a common target cell; said modification domain comprises enzymatic activity exogenous to said target cell; said selectively modifiable activation domain comprises a substrate for said modification domain; and modification of said selectively modifiable activation domain by said modification domain results in proteolytic cleavage and activation of said toxin domain. 